Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD 421, Appel (2004)

As no maternal toxicity or reproductive effects in the females of the satellite groups after mating with the male animals of the main 13-wk study were observed at the high-dose level of this study, this dose level 7500 mg/kg diet (equivalent to 521 mg/ kg body weight/day in males and 433-685 mg/kg body weight/day for females) can be considered as a NOAEL for maternal toxicity and fertility and developmental effects.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 April 2003 to 07 June 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
but none that affected the study validity
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Species: rats
- Strain: Wistar (Crl:(WI)WU BR)
- Age at study initiation: 13-14 weeks old females in the satellite repro study (approximately 17 weeks old males from the main 13-week study, after 10 weeks of treatment)
- Weight at study initiation: body weights for females in the satellite groups at the start of treatment ranged from 194.2 - 229.6 g (mean 208.5 g)
- Feed and drinking water were provided ad libitum.
- Number of animals: 
Dose-range finding study: 21 males and 21 females; 20 of each sex selected (4/sex/group)
Main study (13-week subchronic study) - 43 males and 43 females (4 dose groups of 10 rats/sex/dose group)
Satellite study (reproduction/developmental toxicity screening): 44 females (4 dose groups of 10 females/dose group)
- Acclimation: Upon arrival animals were put in a quarantine room and were given time to acclimate to the new surroundings. The quarantine and acclimatisation periods between arrival and experimental start date were 9, 13, and 13 days for the dose-range finding study, 13-week study, and satellite repro study, respectively.
- Housing: The animals were housed in one room, in macrolon cages, with sterilised wood shavings as bedding material. 2 (dose-range finding study) or 5 rats (13-week study) were housed per cage, separated by sex. During the premating period, females of the satellite groups were housed 3 or 4 per dose group per cage. During gestation and lactation, the females were housed individually.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30-70 %
- Air changes: 10 air changes per hour
- Photoperiod: 12 hr light cycle.
Route of administration:
oral: feed
Vehicle:
other: plain diet
Details on exposure:
ADMINISTRATION / EXPOSURE:
- Duration of exposure: 14 days or 13 consecutive weeks, dose-range finding study and subchronic study (from which the males were used for mating, after 10 weeks of treatment), respectively; females in the satellite reproductive-developmental screening study were exposed for about 5 weeks.
- Type of exposure: via the diet
- Doses: 0, 300, 1500 and 7500 mg/kg diet for the main subchronic study and satellite (reproductive/developmental) study
Details on mating procedure:
- During the mating period (weeks 10 and 11), one male from the 13-week main study and one female of the satellite group of the same dose group were caged until copulation occurred or two weeks had elapsed.
- Every morning vaginal smears were made to ascertain copulation by detection of sperms.
- The day a smear was sperm-positive was considered gestation day 0.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The analytical method was validated in the matrix under examination, viz. RM3 diet, before the start of the study. The test material and other organotin chlorides used as internal standards were converted into the corresponding ethylated tetraorganotin derivatives, which were extracted into the hexane layer. GC-MS analysis was then performed.
- GC-MS was used to determine the achieved concentration, homogeneous distribution and stability of the test material in diet samples. The method of analysis involved derivatisation. This method only measures the amount of the alkyltin moiety, MBT, present and does not identify the other ligands attached to the tin. All measured MBT was attributed to the parent substance, the test material.
- The homogeneity, stability, and achieved concentration (content) of the test material in RM3 rat feed was analysed in the batch of diets prepared for the dose-range finding study and the 13-week study.
- From these analyses, it was concluded that the test material was homogenously distributed in all diets, the test material was stable in the diets at room temperature for 7 days and in the freezer at <-18 °C for 6 weeks, and the content of the test material was close to the nominal level for all diets.
Duration of treatment / exposure:
Dose-range finding study: 14 days
Males derived from the main subchronic 13-week study were treated for 10 weeks before mating with females from the satellite groups around 17 weeks of age. They then continued treatment to complete the 13-week study.
Females in the satellite study were treated via the diet starting 2 weeks before mating, then during mating (up to 2 weeks), gestation, and up to euthanasia at or shortly after postnatal day 4 (ca. 6 weeks).
Frequency of treatment:
Daily
Details on study schedule:
Duration of test: About 6-8 weeks for females in the satellite study (2-week pre-mating, up to 2 weeks for mating, gestation and up to euthanasia at around PND 4).
Dose / conc.:
300 mg/kg diet
Dose / conc.:
1 500 mg/kg diet
Dose / conc.:
7 500 mg/kg diet
No. of animals per sex per dose:
Dose-range finding study: 4 rats/sex/dose group; 5 dose groups including controls
13-week main study and satellite study: 10 rats/sex/dose group and 10 females/dose group, respectively; 4 dose groups including controls
Control animals:
yes, plain diet
Details on study design:
During the premating period females were housed 3 or 4 per group per cage. Male rats of the 13 week study were mated after a premating period of 10 weeks with the satellite female rats.  
During the gestation and lactation periods the females were housed individually. If a male died before or during the mating period before the female was found sperm positive, the female was mated with another, proven male of the same group (i.e. a male which already had a successful copulation, sperm positive smear with another female).
Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS AND FREQUENCY
- Clinical signs: at least once daily (in the morning) and on working days also once in the afternoon.
- Mortality: at least once daily (in the morning) and on working days also at least once in the afternoon.
- Body weight: once during the acclimatisation period, once at initiation of the study prior to introduction of feed and once weekly thereafter. The females of the satellite groups were weighed 4 days before start of dosing at randomisation, on gestation days 0, 7, 14 and 21 and on postnatal days 1 and 4. Furthermore, all surviving animals were weighed on the day of necropsy in order to determine their correct organ to body weight ratios.
- Food consumption: measured per cage over weekly periods during premating and gestation period and from PN 1-4 by weighing the feeders (in g/animal/day and g/kg bw).
- Water consumption: provided ad libitum, the amount consumed was not measured.
- Intake of test material: the intake of test material per kg bw/day was calculated from the nominal dietary concentration of the test material, the food consumption and the mean body weight in the period for which the intake of the test material is calculated.
- Mating: during the mating period every consecutive morning vaginal smears were made to ascertain copulation by detection of sperm cells in the smear.
Oestrous cyclicity (parental animals):
no
Sperm parameters (parental animals):
no
Litter observations:
- Parturition and litter evaluation: at the end of the gestation period, females were examined twice daily for signs of parturition. Any difficulties occurring during parturition were recorded. To keep nest disturbance to a minimum, the litters were examined only once daily for dead pups.
- Litter size, sexes and weights: the total litter size and numbers of each sex as well as the number of stillbirths, live and dead pups and grossly malformed pups were evaluated on days 1 and 4 of lactation. The pups were weighed individually and litter weight was calculated for days 1 and 4 of lactation. Mean pup weights were calculated as litter weight/number pups. The number of runts (pup weight < 2 sd from the control litter mean) were noted and reported as well.
Postmortem examinations (parental animals):
- Macroscopic: all adult animals were subjected to a complete gross necropsy. Organs weighed ifor satellite repro females included: ovaries, uterus (after counting of the implantation sites), thymus and all gross lesions. Samples of latter organs were preserved for microscopic examination.  
- Microscopic: microscopic examination of the ovaries, uterus and thymus of the control and 7500 mg/kg groups was performed. Examination was extended to the thymus of the females of the 300 and 1500 mg/kg groups because of the effects observed in the 7500 mg/kg group for this tissue/organ. Furthermore, the reproductive organs of the males of the 300 and 1500 mg/kg groups that failed to sire (did not mate or female was not pregnant) and the reproductive organs of females of the 300 and 1500 mg/kg groups that were non-mated or non-pregnant were microscopically examined.
Postmortem examinations (offspring):
Necropsy was performed on stillborn pups and pups dying during the study; macroscopic abnormalities were recorded. Pups were examined externally for gross abnormalities and killed by hypothermia at < -18 °C.
Statistics:
- Test material analysis:
Homogeneity: one way analysis of variance (Anova) using the sample location (1-5) as grouping factor. The test material was considered to be homogeneously distributed in the diets if p > 0.01 and/or if the relative standard deviation (RSD) between the samples means was less than or equal to 15 %.
Stability: one way analysis of variance (Anova) using time as grouping factor. The test material was considered to be stable in the diets if p > 0.01 and/or if the mean concentration on the last day was between 80 and 120 % of the mean concentration on the first day (t =0).
Achieved concentration: for each concentration level, the mean of the concentrations, as measured in the diet samples used for the assessment of the homogeneity, was considered to represent the achieved concentration. The content of the test material in the diet was considered to be 'close to intended' if the mean measured concentration was between 80 and 120 % of the intended concentration.
- Body weight: one way analysis of covariance (covariate: body weight on day 0) followed by Dunnett's multiple comparison tests.
- Food consumption and food efficiency: one way analysis of variance (Anova) followed by L.S.D. tests.
- Fisher's exact probability test was used to evaluate the number of mated and pregnant females and females with live pups. Number of implantation sites, live and dead pups were evaluated by Kruskal-Wallis nonparametric analysis of variance followed by the Mann-Whitney U-test.
- Histopathological changes: Fisher's exact probability test.
The litter was used as the statistical unit for calculations of foetal values.
All tests were two-sided. Probability values of p<0.05 were considered significant.
Reproductive indices:
With regard to fertility and reproductive performance, the following parameters were calculated:
- pre-coital time = time between the start of mating and successful copulation
- duration of gestation = time between gestation day 0 and day of delivery
- mating index = (number of females mated/number of females placed with males) x 100
- male fertility index = (number of males that became sires/number of males placed with females) x 100
- female fertility index = (number of pregnant females/number of females placed with males) x 100
- female fecundity index = (number of pregnant females/number of females mated) x 100
- gestation index = (number of females with live pups/number of females pregnant) x 100
- live birth index = (number of pups born alive/number of pups born) x 100
Offspring viability indices:
- pup mortality day n = (number of dead pups on day n/total number of  pups on day n) x 100
- viability index day 1-4 = (number of pups surviving 4 days/total number  of live pups on day 1) x 100
- sex ratio day n = (number of live male pups on day n/ number of live pups on day n) x 100
- number of lost implantations = number of implantations sites - number  of pups born alive
- post-implantation loss = [(number of implantation sites - number of pups born alive)/number of implantation sites] x 100 
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment related clinical signs were observed during the premating, mating, gestation and lactation periods.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality occurred.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- Mean body weight of the females of the 7500 mg/kg group was statistically significantly increased on GD 21 and body weight change was also statistically significantly increased from GD 14-21. During the premating, gestation and lactation periods, no other significant differences in mean body weights and body weight changes of the females were observed.
- Mean food consumption of the females (in g/animal/day and g/kg bw/day) of the 7500 mg/kg group was statistically significantly increased from GD 14-21. During the premating, gestation and lactation periods, no other significant differences in mean food consumption of the females were observed.

Please refer to "Any other information on results incl. tables" for further information
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The test material intake of the female animals of the 300, 1500 and 7500 mg/kg groups was respectively:
Premating period
days 0-7: 19.1, 92.2 and 433.4 mg/kg bw/day
days 7-14: 18.7, 91.4 and 476.4 mg/kg bw/day
Gestation period
GD 0-7: 21.7, 101.8 and 526.2 mg/kg bw/day
GD 7-14: 21.0, 96.5 and 544.0 mg/kg bw/day
GD 14-21: 15.2, 77.3 and 494.3 mg/kg bw/day
Lactation period PN 1-4: 25.3, 140.8 and 684.7 mg/kg bw/day
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
- Reproductive organs and the thymuses of the control and 7500 mg/kg dose groups were examined microscopically. In addition, reproductive organs of the 300 and 1500 mg/kg groups of non-pregnant females and males that failed to sire were examined.
- All histopathological changes in the testes, epididymides, prostate and in the ovaries, uterus and thymuses represent common findings in rats of this strain and age. Moreover, they occurred only incidentally or at similar incidences amongst the groups, including controls. Therefore, they were not considered to be related to treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Analysis of the test material in diet samples revealed that the test material dose was close to the nominal level for all diets.
Homogeneity: The test material was considered to be homogeneously distributed in all diets.
Stability: The test material was considered to be stable in the diets upon storage at room temperature for 7 days and in the freezer at < -18 °C for 6 weeks.
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
- Pre-coital time: comparable among the control and treated groups.
- Mating index: 90 - 100 %
- Number pregnant per dose level: 6, 6, 7 and 7 for the control, 300, 1500 and 7500 mg/kg groups, respectively.
- Female fecundity index: comparable among the control and treated groups.
- Female fertility index: comparable among the control and treated groups.
- Male fertility index: comparable among the control and treated groups.
- Number of implantations: 11-13 (control group), 10-14 (300 mg/kg), 11-14 (1500 mg/kg), 11-13 (7500 mg/kg).
- Gestation index: 100 % in the control, 300, 1500 and 7500 mg/kg groups.
Key result
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
7 500 mg/kg diet
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No significant health effects were observed at the highest dose tested. This dietary dose level is equivalent to 433-685 mg/kg bw/day for females.
Key result
Dose descriptor:
NOAEL
Remarks:
fertility and reproduction
Effect level:
7 500 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean pup weight on PN 1 and 4 of the 7500 mg/kg group was statistically significantly increased. Pup weight change of the pups from the 7500 mg/kg group between PN 1-4 was not statistically significantly increased. No effect on pup weight and pup weight change was observed in the other treated groups when compared to the control group.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
The incidence of runts in the 1500 mg/kg group was statistically significantly increased on PN 1; this observation was only significant on pup basis. No statistically significant difference was observed between the other treated groups and the control group. Macroscopic observation of the stillborn pups revealed no abnormalities of the pups of the 300 and 1500 mg/kg dose groups. The stillborn pup of the 7500 mg/kg group was partly cannibalised; as far as possible no abnormalities were observed in this pup.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- Litter size: The mean number of pups delivered per litter amounted to 8.7, 10.8, 11.0 and 11.3 for the control, 300, 1500 and 7500 mg/kg groups, respectively.
- Live birth index: 100, 100, 100 and 96 % in the control, 300, 1500 and 7500 mg/kg groups, respectively.
- Number of females with live born pups: 6, 6, 7 and 7 in the control, 300, 1500 and 7500 mg/kg groups, respectively.
- Number of females with stillborn pups: 0, 0, 0 and 3 for the control, 300, 1500 and 7500 mg/kg groups, respectively.
- Number of females with all stillborn pups: 0 for the control and all treated groups.
- Post implantation loss: 29.1, 11.4, 17.2 and 10.7 % for the control, 300, 1500 and 7500 mg/kg
- Pup mortality: 0, 0, 0 and 3.8 % for the control, 300, 1500 and 7500 mg/kg groups, respectively (PN 1)
- Number viable: The viability index (PN 1-4) was 100 % in the control, 300, 1500 and 7500 mg/kg groups.
- Number of live pups per litter: 8.7, 10.8, 11.0 and 10.9 for the control, 300, 1500 and 7500 mg/kg groups, respectively (PN 1); 8.7, 10.8, 10.1 and 10.9 for the control, 300, 1500 and 7500 mg/kg groups, respectively (PN 4).
- Sex ratio: No difference was observed in the sex ratio between the groups.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
7 500 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects on pups up to and including lactation day 4
Reproductive effects observed:
no

Among the available studies for the substance investigating pre-natal developmental toxicity in rats, decreased body weight and/or body weight gain in mothers were observed in groups for which toxic effects on mothers were noted. A statistically significant increased mean body weight of females of the high dose group at gestation day (GD) 21, together with statistically significant increases in body weight gain and food consumption for the same group on GD 14-21, was reported in this study and is the only exception.

The above ‘exception’ is apparent due to the greater mean litter size of the high dose females compared to controls.

Of the 10 satellite females per group, 2 control females did not mate and 2 were confirmed as not pregnant. The final number of pregnant females was 6, 6, 7 and 7 in order of ascending dose level. From the table below, it seems that all the 6 females were included in the mean value of the control group whereas 5, 7, and 5 females were included for the treated groups. One female of the low dose and 2 females of the high dose appear missing.

Mean body weights, females (g: satellite groups; gestation period)

 

Group A

control

Group B

300 mg/kg

Group C

1500 mg/kg

Group D

7500 mg/kg

Day 0

213.60 d

220.66

220.80

208.77

Day 7

230.00 d

244.52

237.84

226.83

Day 14

250.55 d

266.98

259.31

249.84

Day 21

283.43 d

300.28

287.74

310.46 *

Statistical key:
d = ANOVA & Dunnett test
* p < 0.05

Individual data reveal that body weight for the missing female of the low dose (No. 131) is not included because it was misjudged to be not mated, while body weights for the 2 missing females of the high dose (No.s 165 and 169) were not recorded at GD 21 because they started delivering in that day. These same females were also not included for the calculation of mean food consumption over GD 14-21.

Individual body weights (g) of rats of the satellite groups (gestation period), Group B: 300 mg/kg

Day of gestation

Female

0

7

14

21

121

213.4

233.7

252.5

285.0

123

217.2

236.6

261.0

282.4

125x  NP

185.6

211.8

212.5

216.1

127

216.7

234.8

262.2

293.0

129x  NP

217.3

241.7

236.5

240.7

131

nm-12

nm-12

nm-12

nm-12

133x  NP

209.9

233.5

230.6

242.8

135

223.0

259.5

274.2

307.0

137x  NP

194.0

217.3

213.8

218.0

139

233.0

258.0

285.0

334.0

Mean

220.66

244.52

266.98

300.28

 

NP: not pregnant
x: excluded from mean
nm-12 : not measured because female was misjudged to be not mated

Individual body weights (g) of rats of the satellite groups (gestation period), Group D: 7500 mg/kg

Day of gestation

Female

0

7

14

21

161

199.2

215.6

235.3

293.4

163x  NP

216.7

219.2

222.7

221.2

165

nm-2

224.4

247.8

nm-12

167x  NP

208.5

233.5

232.8

237.2

169

214.7

241.5

266.4

nm-12

171

204.9

220.4

245.7

314.8

173

207.3

223.1

249.7

304.7

175

208.5

225.7

244.1

311.2

177x  NP

205.7

228.8

232.9

231.3

179

218.0

237.1

259.9

328.2

Mean

208.77

226.83

249.84

310.46

NP: not pregnant
x: excluded from mean
nm-2 : erroneously not measured/ recorded
nm-12 : not measured because female was misjudged to be not mated

In addition to these, although statistical analysis did not show any significant difference between the control and the treated groups, the mean number of pups per litter in the high dose was approximately 30% greater than that of the control group (i.e., 11.29 pups for the high dose vs. 8.67 pups for the control).

Fertility and reproduction

 

Pups delivered (total)

Group A

control

Group B

300 mg/kg

Group C

1500 mg/kg

Group D

7500 mg/kg

Liveborn (N)

52

65

66

79

Liveborn (mean)

8.67

10.83

11.00

11.29

Live Birth Index (%)

100

100

100

96

Stillborn (N)

0

0

0

3

Pup mortality day 1

0.0

0.0

0.0

3.8

 

There were 2 control litters which consisted of only 3 or 4 pups, whereas the lowest number of pups in a litter at the high dose was 9.

Fertility and reproduction (Group A: control)

Litter delivered

Female

Live (N)

Dead (N)

Total (N)

101

11

0

11

103

12

0

12

105  NM

 

 

 

107  NP

 

 

 

109

4

0

4

111

3

0

3

113  NM

 

 

 

115  NP

 

 

 

117

11

0

11

119

11

0

11

NP: not pregnant
NM: not mated

Fertility and reproduction (Group D: 7500 mg/kg)

Litter delivered

Female

Live (N)

Dead (N)

Total (N)

161

11

0

11

163  NP

 

 

 

165 

12

1

13

167   NP 

 

 

 

169

11

1

12

171

11

1

12

173  

10

0

10

175  

9

0

9

177  NP

 

 

 

179

12

0

12

NP: not pregnant

Additionally, group mean body weights for the control and high dose groups on Day 1 of lactation were not statistically different.

Mean body weights, females (g; satellite groups; lactation period)

 

 

Group A

control

Group B

300 mg/kg

Group C

1500 mg/kg

Group D

7500 mg/kg

Day 1

214.27 d

218.28

222.55

229.80

Day 4

230.32 d

236.03

239.74

238.68

d: ANOVA & Dunnett test

It can be concluded that the statistically significant difference in body weight at the end of gestation between the high dose and the control group is real, but has no biological significance other than to reflect litter size and therefore it is not an adverse effect.

Conclusions:
As no maternal toxicity or reproductive effects in the females of the satellite groups after mating with the male animals of the main 13-wk study were observed at the high-dose level of this study, this dose level 7500 mg/kg diet (equivalent to 521 mg/ kg body weight/day in males and 433-685 mg/kg body weight/day for females) can be considered as a NOAEL for maternal toxicity and fertility and developmental effects.
Executive summary:

The toxicity of the test material in Wistar rats was examined using continuous administration via the diet for 13 consecutive weeks in accordance with OECD 408. In satellite groups of female rats a reproduction/developmental screening test was performed in accordance with OECD 421 to provide initial data on the possible reproductive and developmental effects of the test material. The study was performed under GLP conditions.

The main study used four groups of 10 rats/sex (13-week study) and the satellite study used four groups of 10 female rats (reproduction/developmental screening study). For both studies the control group was kept on untreated diet and three test groups received experimental diets containing 300, 1500 and 7500 mg/kg (ppm) of the test material. The dose levels used in both studies were selected based on the results of a preceding dose range finding study.

In the satellite study female rats were fed their respective diets beginning two weeks prior to the mating period, and continued through mating, gestation and up to PN 4 or shortly thereafter. Male rats from the main study were mated after a premating period of 10 weeks with female rats of the satellite groups which were fed the same dose of test diet.

Clinical observations, growth, food consumption, food conversion efficiency, neurobehavioural testing, ophthalmoscopy, haematology, clinical chemistry, renal concentration test, urinalysis, organ weights and gross examination at necropsy, microscopic examination of various organs and tissues and assessment of various reproductive and developmental parameters were used as

criteria for detecting the effects of treatment.

The calculated doses for the females receiving 300, 1500, or 7500 mg/kg test material in the diet ranged between 15.2 -25.3, 77.3-140.8, and 433.4-684.7 mg/kg body weight/day, depending on study period. None of the few changes observed in clinical signs, body weight, food consumption, fertility and reproductive performance, litter data, organ weights, gross macroscopy and microscopic examination were considered treatment-related.

As no maternal toxicity or reproductive effects in the females of the satellite groups after mating with the male animals of the main 13-wk study were observed at the high-dose level of this study, this dose level 7500 mg/kg diet (equivalent to 521 mg/ kg body weight/day in males and 433-685 mg/kg body weight/day for females) can be considered as a NOAEL for maternal toxicity and fertility and developmental effects.

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
other justification
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
The registrant believes that an EOGRTS, with or without cohorts for immunotoxicity and/or neurotoxicity, is not necessary for the registered substance, and that this end point is adequately addressed by the waiver.

In contrast to other organotin substances, which have been shown to possibly possess neurotoxic/ neurodevelopmental and/or immunotoxicity/ developmental immunotoxicity properties, the available data for the registered substance consistently and repeatedly did not provide any evidence for any of these effects.
A fully compliant 90-day repeated dose oral toxicity study performed to OECD 408 that includes a screening study for reproductive toxicity (OECD 421) in rats is available. In this study, male rats from the main groups were treated for 10 weeks (i.e. for the duration of a complete cycle of spermatogenesis) and additional satellite female rats were treated for 2 weeks and then mated. Female rats from the main groups were treated for 13 consecutive weeks.
No adverse effects were observed on fertility, mating behaviour, pregnancy or offspring up to day 4 of lactation. Moreover, no changes in weight or any other findings, including microscopic evaluation of the reproductive tract of animals of either sex were observed.
The only organ showing a change in its weight was the liver (increased at the top dose level). The change in weight was also accompanied by changes in clinical chemistry parameters indicative of liver damage, but there were no microscopic observations.
During the in-life phase, no signs of neurotoxicity were observed in the main group animals. In addition, no significant differences in FOB parameters or at motor activity assessment performed close to the end of the 13-wk treatment period were noted.
No alerts were seen for immunotoxicity. There was no change in absolute or relative weights of thymus and spleen of main group animals, as well as no change in thymus weights of satellite females. Haematology showed an increase of total leukocyte and absolute lymphocyte counts in top dose males, not a decrease.
In addition, several pre-natal developmental toxicity studies in rats are also available for the registered substance, which consistently revealed no effects on pregnancy indices, uterine implantation data, foetal sex ratios, or foetal external, visceral, or skeletal examinations. The maternal and foetal NOAEL values in all these studies were set at the same dose level, indicating no increased sensitivity of the foetus compared to the mothers. The limit dose of 1000 mg/kg/day was set as the developmental LOAEL in the fully compliant key study from Schroeder (2014b), where treatment lasted for the entire pregnancy (gestation days – GD 0-19). In this study, pup weights decreased by 8% compared to controls at 1000 mg/kg/day, only in the presence of maternal toxicity. This manifested as reductions in food consumption (reduced by 9-22% compared to controls) and body weight gain (decreased by 15% compared to controls over GD 0-20, and up to 62% over specific time periods during the treatment). Some wording in the study report is possibly suggestive that some neurological effects were observed in treated females i.e. “The death of one animal in each of the 300 and 1000 mg/kg/day dose groups was not considered test material related. Salivation and audible breathing (stated to be related to salivation afterwards in the report) were observed in the treated groups and considered a pharmacologic response to the test material.” However, the study director did not consider these observations in conjunction with other ‘unusual’ findings at necropsy reported later in the summary (i.e., “A common finding in all the 1000 mg/kg/day animals only at macroscopic examination was white foci in the non-glandular portion of the stomach. Another finding included foreign material generally identified as white in colour and having the appearance of bedding was observed in 7/25 and 23/25 females in the 300 and 1000 mg/kg/day dose groups, respectively, but not observed among the control or 100 mg/kg/day animals. The reason for these animals ingesting the bedding and its toxicological significance in relation to the test material are unclear.”) nor that the registered substance is corrosive to the skin (Cat. 1) and eye (Cat. 1).
In this study, the registered substance was administered by gavage in corn oil. Although homogeneity of the dose preparation was satisfactorily confirmed by analysis, it is unclear if the dose formulation was a solution or suspension, qualities that might (if a suspension) affect distribution of test material onto mucosal surfaces within the GI tract, potentially causing a local irritant effect when given as a bolus dose.
Rather than a pharmacologic response to treatment, salivation and consequent audible breathing can be due to the corrosive properties of the registered substance. In an attempt of relieving the pain in their stomach and the heartburn, animals ingested some bedding material which was found at a high incidence at necropsy in animals of the high and mid dose groups. Additional necropsy findings in the non-glandular stomach also support such hypothesis.
(Please see attached for the relevant results tables from this study)
Based on the available data, and also taking into consideration that the species to be used is the rat, an Extended One-Generation reproductive toxicity study should not be required for the registered substance. None of the above studies indicated any adverse effects on the reproductive performance, organs or tissues, or suggested even possible effects in relation to neurotoxicity and immunotoxicity. A study which can be expected not to provide useful additional information just on the basis of an unproven similarity to other compounds does not appear good use of a large number of additional vertebrates.
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
433 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The quality of the database is high.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Appel (2004)

The toxicity of the test material in Wistar rats was examined using continuous administration via the diet for 13 consecutive weeks in accordance with OECD 408. In satellite groups of female rats a reproduction/developmental screening test was performed in accordance with OECD 421 to provide initial data on the possible reproductive and developmental effects of the test material. The study was performed under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The main study used four groups of 10 rats/sex (13-week study) and the satellite study used four groups of 10 female rats (reproduction/developmental screening study). For both studies the control group was kept on untreated diet and three test groups received experimental diets containing 300, 1 500 and 7 500 mg/kg (ppm) of the test material. The dose levels used in both studies were selected based on the results of a preceding dose range finding study.

In the satellite study female rats were fed their respective diets beginning two weeks prior to the mating period, and continued through mating, gestation and up to PN 4 or shortly thereafter. Male rats from the main study were mated after a premating period of 10 weeks with female rats of the satellite groups which were fed the same dose of test diet.

Clinical observations, growth, food consumption, food conversion efficiency, neurobehavioural testing, ophthalmoscopy, haematology, clinical chemistry, renal concentration test, urinalysis, organ weights and gross examination at necropsy, microscopic examination of various organs and tissues and assessment of various reproductive and developmental parameters were used as criteria for detecting the effects of treatment.

The calculated doses for the females receiving 300, 1500, or 7500 mg/kg test material in the diet ranged between 15.2-25.3, 77.3-140.8, and 433.4-684.7 mg/kg body weight/day, depending on study periods. None of the few changes observed in clinical signs, body weight, food consumption, fertility and reproductive performance, litter data, organ weights, gross macroscopy and microscopic examination were considered treatment-related.

As no maternal toxicity or reproductive effects in the females of the satellite groups after mating with the male animals of the main study were observed at the high-dose level of this study, this dose level of 7500 mg/kg diet (equivalent to 521 mg/ kg body weight/day in males and 433-685 mg/kg body weight/day for females) can be considered as a NOAEL for maternal toxicity and fertility and developmental effects.

Extended One Generation Reproductive Toxicity Study

An EOGRTS, with or without cohorts for immunotoxicity and/or neurotoxicity, is not necessary for the registered substance, and this endpoint is adequately addressed by the following waiver:

"In accordance with Regulation EC 1907/2006 (REACH), Annex IX point 8.7.2 of column 1, an Extended One-Generation reproductive toxicity study should be performed in an appropriate species with regard to the likely route of exposure if the 28-day or 90-day study indicates adverse effects on the reproductive organs or tissues or reveal other concerns in relation with reproductive toxicity. For the registered substance, a 90-day repeated dose oral toxicity performed to OECD 408 and inclusive of a screening for reproductive toxicity (OECD 421) in rats is available. Male rats from the main groups were treated for 10 weeks (i.e. for the duration of a complete cycle of spermatogenesis) and satellite female rats were treated for 2 weeks and then mated. No adverse effects were observed on fertility, mating behaviour, pregnancy or offspring up to day 4 of lactation. Moreover, no changes in weight, or any other findings in reproductive tract were observed. In addition to these repeated-dose studies, several pre-natal developmental toxicity studies in rats are also available for the registered substance. All these consistently revealed no effects on pregnancy indices, uterine implantation data, foetal sex ratios, or foetal external, visceral, or skeletal examinations. The maternal and foetal NOAEL values in all these studies were set at the same dose level, indicating no increased sensitivity of the foetus compared to the mothers. The limit dose of 1000 mg/kg/day was in some instances set as the developmental LOAEL, because of slight reduction in pup weights in the presence of maternal toxicity. Based on the available data, and considering that the preferred species for reproductive/fertility toxicity studies is the rat, an Extended One-Generation reproductive toxicity study is not required for the test material."

In contrast to other organotin substances, which have been shown to possibly possess neurotoxic/ neurodevelopmental and/or immunotoxicity/ developmental immunotoxicity properties, the available data for the registered substance consistently  and repeatedly did not provide any evidence for any of these effects.  

A fully compliant 90-day repeated dose oral toxicity study performed to OECD 408 that includes a screening study for reproductive toxicity (OECD 421) in rats is available. In this study, male rats from the main groups were treated for 10 weeks (i.e. for the duration of a complete cycle of spermatogenesis) and additional satellite female rats were treated for 2 weeks and then mated. Female rats from the main groups were treated for 13 consecutive weeks.

No adverse effects were observed on fertility, mating behaviour, pregnancy or offspring up to day 4 of lactation. Moreover, no changes in weight or any other findings, including microscopic evaluation of the reproductive tract of animals of either sex were observed.

The only organ showing a change in its weight was the liver (increased at the top dose level). The change in weight was also accompanied by changes in clinical chemistry parameters indicative of liver damage, but there were no  microscopic observations.

During the in-life phase, no signs of neurotoxicity were observed in the main group animals. In addition, no significant differences in FOB parameters or at motor activity assessment performed close to the end of the 13 -wk treatment period were noted.

No alerts were seen for immunotoxicity. There was no change in absolute or relative weights of thymus and spleen of main group animals, as well as no change in thymus weights of satellite females. Haematology showed an increase of total leukocyte and absolute lymphocyte counts in top dose males, not a decrease.

In addition, several pre-natal developmental toxicity studies in rats are also available for the registered substance, which consistently revealed no effects on pregnancy indices, uterine implantation data, foetal sex ratios, or foetal external, visceral, or skeletal examinations. The maternal and foetal NOAEL values in all these studies were set at the same dose level, indicating no increased sensitivity of the foetus compared to the mothers. The limit dose of 1000 mg/kg/day was set as the developmental LOAEL in the fully compliant key study from Schroeder (2014b), where treatment lasted for the entire pregnancy (gestation days – GD 0-19). In this study, pup weights decreased by 8% compared to controls at 1000 mg/kg/day, only in the presence of maternal toxicity. This manifested as reductions in food consumption (reduced by 9-22% compared to controls) and body weight gain (decreased by 15% compared to controls over GD 0-20, and up to 62% over specific time periods during the treatment). Some wording in the study report is possibly suggestive that some neurological effects were observed in treated females i.e. “The death of one animal in each of the 300 and 1000 mg/kg/day dose groups was not considered test material related. Salivation and audible breathing (stated to be related to salivation afterwards in the report) were observed in the treated groups and considered a pharmacologic response to the test material.” However, the study director did not consider these observations in conjunction with other ‘unusual’ findings at necropsy reported later in the summary (i.e., “A common finding in all the 1000 mg/kg/day animals only at macroscopic examination was white foci in the non-glandular portion of the stomach. Another finding included foreign material generally identified as white in colour and having the appearance of bedding was observed in 7/25 and 23/25 females in the 300 and 1000 mg/kg/day dose groups, respectively, but not observed among the control or 100 mg/kg/day animals. The reason for these animals ingesting the bedding and its toxicological significance in relation to the test material are unclear.”) nor that the registered substance is corrosive to the skin (Cat. 1) and eye (Cat. 1).

In this study, the registered substance was administered by gavage in corn oil. Although homogeneity of the dose preparation was satisfactorily confirmed by analysis, it is unclear if the dose formulation was a solution or suspension, qualities that might (if a suspension) affect distribution of test material onto mucosal surfaces within the GI tract, potentially causing a local irritant effect when given as a bolus dose.

Rather than a pharmacologic response to treatment, salivation and consequent audible breathing can be due to the corrosive properties of the registered substance. In an attempt of relieving the pain in their stomach and the heartburn, animals ingested some bedding material which was found at a high incidence at necropsy in animals of the high and mid dose groups. Additional necropsy findings in the non-glandular stomach also support such hypothesis.

Based on the available data, and also taking into consideration that the species to be used is the rat, an Extended One-Generation reproductive toxicity study should not be required for the registered substance. None of the above studies indicated any adverse effects on the reproductive performance, organs or tissues, or suggested even possible effects in relation to neurotoxicity and immunotoxicity. A study which can be expected not to provide useful additional information just on the basis of an  unproven similarity to other compounds does not appear good use of a large number of additional vertebrates.

In addition the Registrant proposes to waive the Extended One Generation Reproductive Toxicity Study specified in Section 8.7.3 of Annex X of the Regulation on Substance-Tailored, Exposure-Driven grounds as set out in Section 3 of Annex XI of the Regulation.

DNELs for the registered substance have been derived using the lowest oral NOEL (rather than a NOAEL) value identified among all available animal studies and, for exposure via inhalation, the NOAEL obtained in a 28-day inhalation study. In particular for the oral starting point, rather than choosing the NOAEL of 96 mg/kg/day obtained in a fully compliant OECD 408 subchronic toxicity study, based on effects on liver, or the NOAEL of 433 mg/kg/day for reproduction obtained in the satellite OECD 421 performed concomitantly to the same subchronic study, the NOEL for maternal toxicity obtained in a fully compliant OECD 414 study in rabbits has been used. Further explanations on the chosen starting point, as well as clear demonstrations that the more precautionary approach has been applied, are included in the CSR.

Even using this extremely precautionary approach, the Risk Characterisation Ratios calculated in the Chemical Safety Report are all significantly less than 1.

Existing data on the toxicological properties of the registered substance are extensive, and include an oral subchronic toxicity study in rats (OECD 408) also inclusive of a satellite screening for reproductive and developmental effects (OECD 421), and a subacute 28-day repeat dose toxicity study (with a recovery period) via the inhalation route. Developmental toxicity has also been further evaluated, in prenatal developmental toxicity studies (OECD 414) in two species, i.e. the rat and the rabbit.

Considering all available studies conducted with rats, systemic effects on the liver following oral administration, and unspecific general toxicity following inhalation were at the basis of NOAEL setting. No effects on reproductive performance, and no indications of neurological or immunological effects were observed. In the prenatal developmental toxicity study in rats no teratogenic effects were observed. The only developmental effect was a decrease of pup weights (8% compared to controls) at 1000 mg/kg/day and then only in the presence of maternal toxicity. At the limit dose maternal toxicity manifested as reductions in food consumption (reduced by 9-22% compared to controls) and body weight gain (decreased by 15% compared to controls over GD 0-20, and up to 62% over specific time periods during the treatment period comprising the entire gestation (i.e., GD 0-19).

Results of the prenatal developmental toxicity study in rabbits indicate an increase in adverse pregnancy outcomes (i.e., increase in post-implantation loss, increases in total and early resorptions, and lower foetal bodyweights), secondary to maternal toxicity, essentially observed at the top dose level. At all dose levels, the number of viable litters at term was adequate for evaluating any possible developmental effects and no teratogenic effects were noted. The NOEL for maternal toxicity was set at the lowest dose level tested (30 mg/kg/day) because one female at the mid-dose aborted: this female was clearly more affected by treatment than the other does of the same group.

The species to be used for the EOGRTS is the rat. Available existing robust data in the rat already determined that the registered substance is of no concern for fertility and developmental toxicity in this species, and no suggestion of possible neurotoxic or immunotoxic effects in this species were ever observed.

On this basis the registrant considers that the EOGRTS would not provide any additional useful information, and would just represent an undue, not scientifically sound use of a large number of additional rats.

Effects on developmental toxicity

Description of key information

Two key studies were presented to address this endpoint. Both studies were performed under GLP conditions and in accordance with standardised guidelines US EPA OPPTS 870.3700 and OECD 414. In both studies, the test material was found not to be teratogenic to rats and rabbits. Adverse pregnancy outcomes were observed in rabbits only; however these were concluded to be test material related, but secondary to maternal toxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 June 2014 to 05 September 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Hra:(NZW) SPF
- Age at study initiation: 5 to 5.5 months
- Weight at study initiation: 2.56 to 3.63 kg
- Housing: Individually housed in suspended, stainless steel, slatted floor cages. Animal enrichment was provided.
- Diet: Limited to 50 g/animal/day on arrival (GD 0) and increased on second day of acclimation to approximately 170 g/animal/day for the remainder of the study. Enrichment food was offered on occasion.
- Water: tap water was available ad libitum via an automatic watering system
- Acclimation period: 1 day

ENVIRONMENTAL CONDITIONS
- Temperature: 61-72 °F
- Humidity: 30 to 70 %
- Photoperiod: 12 hour light/dark cycle

IN-LIFE DATES: From: 10 June 2014 To: 11 July 2014
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test material in vehicle was prepared weekly and stored refrigerated at 2 to 8 °C (protected from light).

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected based on information from a pilot study.
- Concentration in vehicle: 60, 120 and 240 mg/mL
- Amount of vehicle (if gavage): 0.5 mg/mL
- Lot/batch no.: 2CK0155
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing samples were evaluated for homogeneity and concentration. 1 mL samples were collected for homogeneity during week 1 from the top, middle and bottom of the 30 and 120 mg/kg formulations. Samples from all concentrations were taken during weeks 1 and 4 from the middle stratum for analysis of concentration. The dose formulations were analysed using HPLC/UV.

HPLC/UV CONDITIONS
Column: Agilent Poroshell 120 SB-C8 column, 3.0 x 50 mm
Gradient flow: 0.05 % trifluoroacetic acid in water (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 1.0 mL/minute.
Wavelength: 230 nm
Derivatisation: Prior to analysis, duplicate samples were derivatised in hexane and phenylmagnesium bromide then diluted with isopropyl alcohol to within the range of the calibration curve. Vehicle samples were also derivitised and then diluted using a dilution factor of 100.

RESULTS
- Homogeneity:
Analysis of the low- and high-dose formulations (60 and 240 mg/mL, respectively) used for dosing in the first week of study confirmed they were homogeneous as prepared, meeting the laboratory’s acceptance criteria of 100 ± 15 % of nominal, and percent relative standard deviation (RSD) ≤10.
- Concentration:
Mean concentrations of formulations used for dosing in Weeks 1 and 4 of study ranged between 96.7 and 104.7 % of nominal with percent RSDs ranging between 0.523 and 2.649, confirming that animals were receiving the appropriate dose levels when the formulation was administered at 0.5 mL/kg. No test material was detected in the control samples.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
Gestation day 1 to 28
Frequency of treatment:
Once daily
Duration of test:
29 days
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
120 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on information from a pilot study.
In the pilot study, dose levels of 30, 65, 140 and 300 mg/kg/day were evaluated. However, due to reduced food consumption levels in several groups by GD 5, the dose levels were adjusted to 30 (unchanged), 50, 100 and 200 mg/kg/day. On GD 8, the 200/300 mg/kg/day dose group was terminated due to continued low food consumption, weight loss and being in a state of in extremis. In all remaining groups there was variability in food consumption as expected with the corn oil vehicle administered at 1 mL/kg/day. One animal died in the 100 mg/kg/day dose group (cause undetermined from necropsy observations) and one animal aborted in each of the 30 and 100 mg/kg/day dose groups. The aborted pregnancies were preceded by lengthy periods of inappetence. The maternal toxicity observed in the pilot study was considered largely attributed to the 1 mL/kg corn oil vehicle treatment and in the main study this volume was reduced to 0.5 mL/kg. No effect of treatment to 100 mg/kg/day was observed on uterine implantation data or foetal external examinations. There was about a 10 % reduction in mean foetal body weight at the 100 mg/kg/day dose level relative to controls.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations: Morbidity, mortality, injury and availability of food and water.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily
- Examinations: Animals were removed from the cage and given a detailed clinical examination. On occasion, clinical observations were recorded at unscheduled intervals. The observations included, but were not limited to, evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, as well as evaluation of respiration.

BODY WEIGHT: Yes
- Time schedule for examinations: Gestation days 0, 1, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 29. Body weight change was calculated for gestation days 0-1, 1-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27, 27-29 and 1-29. Adjusted body weight (minus gravid uterus) and adjusted bodyweight change were also calculated.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: Food consumption recorded daily and reported on the corresponding body weight days.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29. Each surviving female was euthanised by injection with euthanasia solution, followed by exsanguination from the femoral vessels and immediately subjected to a laparohysterectomy.
- Organs examined: Does were subjected to a complete necropsy. Special emphasis was placed on structural abnormalities or pathologic changes that may have influenced pregnancy.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
Statistics:
All parental in-life data, gravid uterine weights, corpora lutea/doe, total implantations/doe, litter size/doe, viable foetuses/doe, total number of resorptions/doe, number of early resorptions/doe and number of late resorptions/doe were assessed using group pair-wise comparisons. Levene's test was used to assess homogeneity of group variances. If Levene's test was not significant (p≥0.01), a pooled estimate of variance (Mean Square Error) was computed from a one-way analysis of variance (ANOVA) and utilised by a Dunnett's comparison of each treatment group with the control. If the Levene's test was significant (p<0.01), then Welch's t-test was used with a Bonferroni correction for comparisons with the control group. All endpoints were analysed using two-tailed tests.
Foetal sex ratio (% males/litter), % preimplantation loss and % postimplantation loss were analysed using Arcsin-Square-Root Transformation. Percent values were transformed using the arcsin of the square root and analysed in accordance with the group pair-wise comparisons.
Pregnancy index, malformations and variations were all evaluated using Fisher’s exact test. An overall test for association between response and treatment was performed. If this test was significant (p<0.05) and there were more than two groups, then each treatment group was compared to the control group. All endpoints were analysed sing two-tailed tests. Overall testing and follow-up pair-wise testing was conducted for the control and all treatment groups that had sufficient sample size (n≥3).
Mean foetal bodyweights were analysed by covariate analysis by comparing treatment groups for each endpoint (mean of foetal bodyweights per female). The covariate was litter size. Each treatment satisfying the sample size assumption was compared to the control using Dunnett's test under the analysis of covariance model. Endpoints were analysed using two tailed tests.
The number of non-viable foetuses/doe was analysed by descriptive statistics.
Indices:
- Viable foetuses
- Postimplantation loss = ((No. implantations - No. viable foetuses) / No. implantations) x 100
- Preimplantation loss = ((No. corpora lutea - No. implantations) / No. corpora lutea) x 100
- Pregnancy index = (No. pregnant females - No. females with evidence of mating) x 100
- Foetal sex ratio
Historical control data:
A large database of historical control data for the rabbit was available for comparison.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- In the 30 and 60 mg/kg bw/day dose groups, no clinical effects were noted at post-dose examination. There was a slight decrease in defecation in the 30 and 60 mg/kg bw/day dose groups. This generally correlated with late gestation inappetence which is common in rabbits and not considered to be test material related.
- In the 120 mg/kg bw/day group, findings attributed to the test material included 2/25 animals with decreased activity, abnormal shaped faeces in 4/25 animals, discoloured urine (orange) in 13/25 animals and thin appearance in 11/25 animals. There was also a decrease in defecation in these animals (19/25 animals) appearing from around gestation day 4 and lasting throughout gestation. The decreased defecation in this group correlated with low food consumption and was considered test material related. Brown/red material was found in the sub-cage pan of 7 animals in the 120 mg/kg bw/day. This was related to failed pregnancies in 5 of these animals (two aborted pregnancies, one early delivery and two litters with resorbing foetuses). No further treatment related clinical findings were observed in the 120 mg/kg bw/day group.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- At all dose levels tested, there were no effects observed on maternal survival. For all animals that died or were euthanised, the cause of death was attributed to dosing injuries, and were not considered to be test material related.
- A single animal in the 30 mg/kg bw/day group died on gestation day 29 prior to euthanasia. The death of this animal was attributed to a dosing event.
- An animal in the 60 mg/kg bw/day group was noted to have difficulty breathing on gestation day 6 and was euthanised in extremis; this was also attributed to a dosing event and was not considered test material related.
- An animal in the 120 mg/kg bw/day group was found dead on gestation day 24. Apart from being noted as thin on gestation day 3, the animal was found to be clinically unremarkable thereafter. Findings at necropsy indicated that this was also the result of a dosing event.
- All other animals survived until scheduled sacrifice.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- No adverse effects on bodyweight and bodyweight change were noted at 30 and 60 mg/kg bw/day.
- In the 120 mg/kg bw/day group, mean bodyweights from gestation day 6 to 29 were 5 to 7 % lower than controls and were found to be statistically significant. Statistically significant differences in bodyweight change were noted in this group when compared to controls over gestation days 3 to 6 and over the entire gestation period. These observed differences correlated with reduced food consumption and were attributed to test material administration.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- No adverse effects were observed at 30 and 60 mg/kg bw/day on food consumption. Throughout the entirety of gestation, food consumption in the 30 mg/kg bw/day group was found to be comparable to controls and in the 60 mg/kg bw/day group with the exception of gestation days 27 to 29. As food consumption in the 60 mg/kg bw/day group was comparable to the control group for gestation as a whole and in the absence of an effect on food consumption earlier in gestation, this was not considered to be a toxicological response to test material administration.
- In the 120 mg/kg bw/day group, the mean food consumption was 17 to 54 % lower than control values for the majority of the intervals recorded. Most of these differences were found to be statistically significant, and overall throughout the entirety of gestation food consumption was found to be 41 % lower than controls in this group. The lower food consumption in this group correlated with effects noted on bodyweight and bodyweight change and was determined to be test material related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
- At necropsy, no effect of treatment was observed at any dose level tested. The findings observed were found to be of low incidence and not related to test material administration.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
- One animal in the 60 mg/kg bw/day group and two animals in the 120 mg/kg bw/day group aborted.
Abortion can occasionally occur in rabbits, and a single abortion at the mid dose is within normal variability; this is also supported by the Historical control data included in the report. However, body weight and food consumption data for this specific mid dose female (No. 257) which aborted indicate signs of toxicity (reduced food consumption and body weight loss).
The two females of the high dose group that aborted (No. 287 and No. 281) also showed significant deficits of food consumption and weight gain, even relative to the group mean values for the top dose group.
Consistent with the results of the dose ranging study, the principal maternal effect was a reduction in food consumption; a clear treatment-related effect is evident on both food consumption and group mean body weight at the top dose level and, to a lesser extent, for the single female of the mid dose group which aborted. Maternal well-being was insufficient to maintain pregnancy.
In summary, all abortions were preceded by signs of toxicity (clinical signs, low weight gain or weight loss, and extended periods of inappetence) and were considered to be related to treatment with the test material, but secondary to maternal toxicity and not due to a direct effect of the test substance.
Exclusive of these comprised pregnancies, there were 25, 23, 23, and 18 GD 29 pregnancies with viable fetuses for evaluation of developmental toxicity in the control, 30, 60, and 120 mg/kg/day dose groups, respectively.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
- There were two females in the 120 mg/kg bw/day group with only resorbing foetuses in utero (100 % post implantation loss).
In the 120 mg/kg bw/day group, an increase in post-implantation loss was recorded. The mean post-implantation loss was higher at 15.32 % compared to 2.09 % in controls, though this was not found to be statistically significant. When these two females with only resorbing fetuses are excluded from the calculation of the group mean, there is no clear increase.
The increase in post-implantation loss was considered to be secondary to maternal toxicity evidenced through significant deficits of food consumption and weight gain in affected animals.
- At 30 and 60 mg/kg bw/day, no effect was noted on uterine implantation.
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
- There were two females in the 120 mg/kg bw/day group with only resorbing foetuses in utero (100 % post implantation loss).
In the 120 mg/kg bw/day group, the mean number of early resorptions and total resorptions were statistically higher, 1.3 and 1.4, respectively, when compared to control values of 0.1 and 0.2, respectively. This increase was considered to be secondary to maternal toxicity evidenced through significant deficits of food consumption and weight gain in affected animals.
- At 30 and 60 mg/kg bw/day, no effect was noted on uterine implantation.
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
- At 30 and 60 mg/kg bw/day, no effect was noted on uterine implantation. In the 120 mg/kg bw/day group, the mean number of early resorptions and total resorptions were statistically higher, 1.3 and 1.4, respectively, when compared to control values of 0.1 and 0.2, respectively. This increase was considered to be secondary to maternal toxicity evidenced through significant deficits of food consumption and weight gain in affected animals.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
effects observed, treatment-related
Description (incidence and severity):
One animal in the 120 mg/kg bw/day group delivered early. This animal showed marked toxicity in this study. The litter of 7 kits appears otherwise normal from the report and accounted for all implantation sites; no effect on development can be inferred. Early delivery, even if plausibly treatment-related, however is insufficient evidence for classification in this study since it is confined to a single animal.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Pregnancy indices were 100, 96, 100 and 96 % in the control, 30, 60 and 120 mg/kg bw/day groups, respectively.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Mean gravid uterine weights and adjusted weight change in the low and mid dose groups were comparable to the control group. In the 120 mg/kg bw/day group, the mean adjusted weight change over the gestation period was statistically significant as an overall loss of 0.137 kg was observed compared to a 0.039 kg weight gain in controls. The mean gravid uterine weight and adjusted bodyweight in this group were comparable to mean control values.
Key result
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
number of abortions
pre and post implantation loss
total litter losses by resorption
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
- No effect of the test material was observed on foetal bodyweight with administration of the test material at 30 and 60 mg/kg bw/day to the mothers.
- In the 120 mg/kg/day dose group, mean foetal weights, distinguished by sex and for the combined sexes were 11 to 13 % lower than mean control values. These however mirrored maternal weight change, and can be attributed to the maternal weight effect.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No effect was observed on foetal sex ratio with the administration of the test material at any dose level. These mean ratios ranged from 49.9% to 53.4% in the treated groups and were comparable to the 51.2% in controls.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No effects were observed in litter size, at any dose level. Litter weight was not determined.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- No effect of treatment was observed at all dose levels evaluated with respect to external examinations.
- The external malformations observed in the mid and high dose group foetuses were dissimilar, occurred at low incidence (single foetus affected) or with similar frequency as the controls. These were therefore considered to be unrelated to administration of the test material.
- No external malformations were observed in the 30 mg/kg bw/day group.
- No external developmental variations were observed among foetuses in the control or treated groups.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- No effect of treatment was observed in the skeletal examinations at all dose levels tested.
In the 60 mg/kg bw/day group, the litter incidence of unossified sternebrae, a common developmental variation, was 73.9 %; this was statistically different from the 28 % observed in controls, and just outside the maximum incidence commonly observed in the historical control data. With the absence of a similar increase in incidence of this variation in the 120 mg/kg bw/day group (50%), this was considered to be an incidental occurrence and not test material related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- No effect of treatment at the dose levels evaluated was observed from the foetal visceral examinations.
- The few visceral malformations and developmental variations observed in the treated groups occurred at low incidence or with similar frequency as in controls and were considered unrelated to the test material.
Other effects:
not examined
Key result
Dose descriptor:
NOEL
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduced foetal weight
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
120 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes

Results of the study indicate an increase in adverse pregnancy outcomes (secondary to maternal toxicity), an increase in post-implantation loss, increases in total and early resorptions, and lower foetal bodyweights at the top dose level. At all dose levels, the number of viable litters at term was adequate for evaluating any possible developmental effects; no teratogenic effects were noted.

A summary of pregnancy outcomes is presented here below.

Key pregnancy outcomes with individual animal identifiers

 

Dose, mg/kg bw/day

0

30

60

120

Number of females

25

25

25

25

Non-pregnant

0

1

0

1

Died or euthanized

0

1 (247)

1 (252)

1 (282)

Abortion

0

0

1 (257)

2 (278, 281)

Early delivery

0

0

0

1 (288)

Litters entirely resorbed in utero

0

0

0

2 (280, 292)

Viable litters at term

25

24

23

18

Individual animal ID numbers of affected rabbits given in parentheses

 

For all animals that died or were euthanized, the cause of death was attributed to dosing injuries, unrelated to test material administration.

Abortion can occasionally occur in rabbits, and a single abortion at the mid dose is within normal variability; this is also supported by the Historical control data included in the report.

Historical control developmental toxicity data New Zealand White rabbit (test facility 08.01.2009 to 08.01.2014)

 

Number of studies

32

Number of females

701

Number pregnant

650

Number not pregnant

51

Number that died or pregnant euthanised moribund

4

Number of abortions

2

Number of early deliveries

3

Number with all resorptions

2

Number pregnant by stain

0

Number of females with viable fetuses day 29 gestation

639

 

Overall, the mid dose mothers did not show any signs of toxicity, and no significant difference from the control group, particularly for effects on food consumption and/or body weight were recorded. Body weight and food consumption data for the mid dose female (No. 257) which aborted however indicate severe effects, possibly related to treatment.

A comparison of rabbit food consumption and weight gain in all unsuccessful pregnancies in the study is presented in the table below.


Comparison of rabbit food consumption and weight gain in unsuccessful pregnancies (Schroeder 2014a)

 

 

Study Period (days)

 

 

0

1

3

6

9

12

15

18

21

24

27

29

Control group mean

BW

3.13

3.19

3.25

3. 31

3.36

3.40

3.50

3.52

3.55

3.60

3.63

3.65

BW gain

 

60

60

60

50

40

100

20

30

50

30

20

FC

 

52.04

150.76

156.20

149.92

147.13

132.65

142.91

142.44

111.73

93.23

88.22

Mid dose group mean

BW

3.11

3.15

3.21

3.28

3.33

3.39

3.48

3.52

3.54

3.58

3.59

3.60

BW gain

 

40

60

80

30

50

100

30

30

40

20

-10

FC

 

53.04

143.04

147.16

137.32

140.54

122.61

136.44

135.56

110.54

80.69

50.54*

High dose group mean

BW

3.13

3.15

3.17

3.15*

3.16**

3.18**

3.26**

3.26**

3.31**

3.36**

3.40*

3.42*

BW gain

 

30

20

-20**

10

10

80

0

60

50

30

-10

FC

 

49.75

109.29**

90.65**

71.67**

67.02**

63.58**

70.64**

88.00**

91.40

77.41

59.00

Individual values

257 – aborted (mid dose)

BW

3.25

3.28

3.29

3.38

3.32

3.38

3.34

3.31

3.23

3.22

3.24

NDC

BW gain

 

30

10

90

-60

60

-40

-30

-80

-10

20

NDC

FC

 

55.0

125.5

140.7

77.0

84.7

10.0

3.7

11.7

25.3

34.0

NDC

278 – aborted

BW

3.47

3.58

3.51

3.38

3.27

3.26

3.28

3.34

3.33

3.25

NDC

 

BW gain

 

110

-70

-130

-110

-10

20

60

-10

-80

NDC

 

FC

 

50.0

134.0

40.7

0.7

6.0

0.7

0.3

0.3

0.7

0.0

NDC

280 – all resorbed

BW

3.24

3.24

3.27

3.17

3.14

2.98

2.97

2.88

2.76

2.99

3.11

3.08

BW gain

 

0

30

-100

-30

-160

-10

-90

-120

230

120

-30

FC

 

36.0

114.0

60.7

0.3

5.3

1.0

2.0

2.3

127.0

155.0

97.5

281 - aborted

BW

3.31

3.39

3.33

3.21

3.15

3.11

3.15

3.00

3.25

NDC

 

 

BW gain

 

80

-60

-120

-60

-40

40

-150

250

NDC

 

 

FC

 

47.0

35.0

18.7

2.5

3.5

0.0

0.0

33.7

0.0

NDC

 

288 – premature delivery

BW

2.58

2.65

2.63

2.73

2.77

2.74

2.79

2.73

2.74

2.74

2.83

NDC

BW gain

 

70

-20

100

40

-30

50

-60

10

0

90

NDC

FC

 

59.0

103.5

104.3

84.0

71.3

21.3

2.7

1.7

1.3

4.0

NDC

292 – all resorbed

BW

3.16

3.10

3.17

3.15

3.06

2.96

3.18

3.35

3.35

3.41

3.50

3.52

BW gain

 

-60

70

-20

-90

-100

220

170

0

60

90

20

FC

 

30.0

105.5

66.7

0.0

16.0

100.0

107.0

108.3

123.0

169.3

152.5

 

BW: bodyweight (kg); BW gain: body weight gain (g) (presented in kg in the report); FC: food consumption (g/animal/day during the period between bodyweight measurement). Values in bold show clear deficits from normal.

NDC: no data collected. Level of statistical significance: * p >0.05, ** p> 0.01 in comparison with control.


The above data clearly demonstrate that all the affected animals showed significant deficits of food consumption and weight gain, even relative to the group mean values for the top dose group. Consistent with the results of the dose ranging study, the principal maternal effect was a reduction in food consumption; a clear treatment-related effect is evident on both food consumption and group mean body weight at the top dose level and, to a lesser extent, for the single female of the mid dose group which aborted. Maternal well-being was insufficient to maintain pregnancy.

As mentioned above, developmental effects were all considered secondary to maternal toxicity and consisted in an increase in post-implantation loss, statistically higher mean number of early and total resorptions and increased, although not statistically significant, mean post-implantation loss at 120 mg/kg/day. All other uterine implantation parameters (corpora lutea, implantation sites, preimplantation loss, viable fetuses, nonviable fetus, and litter size) were comparable to mean control values.

In addition, mean fetal weights, distinguished by sex and for the combined sexes were 11% to 13% lower than mean control values at 120 mg/kg/day.

Endpoint

0

30

60

120

Number of females on study

25

25

25

25

Non-pregnant

0

1

0

1

Number pregnant

25

24

25

24

Pregnancy index percentage

100.0

96.0

100.0

96.0

Number died/ euthanised moribund

0

1

1

1

Number of abortions

0

0

1

2

Number of early deliveries

0

0

0

1

Number of females with all resorptions

0

0

0

2

Number of females with viable fetuses (day 29 gestation)

25

23

23

18

Corpora lutea (mean per animal)

9.5

10.0

9.9

8.8

Implantation sites (mean per animal)

8.6

8.9

9.1

9.1

Preimplantation loss (mean per animal), %

9.04

11.31

7.63

5.64

Viable fetuses (mean per animal)

8.4

8.7

8.9

7.8

Fetal sex ratio (mean % males per animal)

51.2

53.4

51.8

49.9

Postimplantation loss (mean per animal), %

2.09

4.61

3.06

15.32

Nonviable fetuses (mean per animal)

0.0

0.0

0.0

0.0

Litter size (mean per animal)

8.4

8.7

8.9

8.6

Resoprtions (early + late) (mean per animal)

0.2

0.3

0.3

1.4*

Early resorptions (mean per animal)

0.1

0.1

0.2

1.3*

Late resorptions (mean per animal)

0.1

0.1

0.0

0.1

 * p >0.05

The “increased post-implantation loss” was attributable to two top dose litters showing complete resorption, in turn a consequence of marked maternal toxicity. When these two values are excluded from the calculation of the group mean, there is no clear increase.

Dose, mg/kg bw/day

0

30

60

120

Post implantation loss, % (report p64)

2.09

4.61

3.06

15.32

Excluding 2 litters with all resorptions

 

 

 

6.6

 

The “lower foetal weights” were significant at the high dose only. These however mirrored maternal weight change, and can be attributed to the maternal weight effect.

Dose, mg/kg bw/day

0

30

60

120

Final maternal bodyweight, mean (kg)

3.6

3.6

3.6

3.4**

Maternal adjusted weight gain (g)

40

30

10

-130*

Foetal bodyweight, mean (g)

40.0

40.0

37.9

35.6**

Level of statistical significance: * p >0.05, ** p > 0.01 in comparison with control

 

There were no teratogenic effects, and no dose-related increase in the number of variants either on a fetal or litter basis, at external, visceral and skeletal examinations.

Summary of external malformations and developmental variations

Observation

0

30

60

120

Number of litters evaluated

25

24

23

18

Number of fetuses evaluated

211

206

204

155

Total malformations - number of litters (%)

1 (4.0)

0 (0.0)

2 (8.7)

1 (5.6)

Total malformations - number of fetuses (%)

1 (0.5)

0 (0.0)

2 (1.0)

1 (0.6)

Total variations - number of litters (%)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

Total variations - number of fetuses (%)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

 

Summary of visceral malformations and developmental variations

Observation

0

30

60

120

Number of litters evaluated

25

24

23

18

Number of fetuses evaluated

211

206

204

155

Total malformations - number of litters (%)

2 (8.0)

1 (4.2)

1 (4.3)

1 (5.6)

Total malformations - number of fetuses (%)

2 (0.9)

1 (0.5)

1 (0.5)

1 (0.6)

Total variations - number of litters (%)

10 (40.0)

3 (12.5)

6 (26.1)

5 (27.8)

Total variations - number of fetuses (%)

14 (6.6)

5 (2.4)

8 (3.9)

9 (5.8)

 

Summary of skeletal malformations and developmental variations

Observation

0

30

60

120

Number of litters evaluated

25

24

23

18

Number of fetuses evaluated

211

206

204

155

Total malformations - number of litters (%)

10 (40.0)

6 (25.0)

2 (8.7)

3 (16.7)

Total malformations - number of fetuses (%)

12 (5.7)

8 (3.9)

2 (1.0)

3 (1.9)

Total variations - number of litters (%)

23 (92.0)

23 (95.8)

23 (100.0)

15 (83.3)

Total variations - number of fetuses (%)

101 (47.9)

85 (41.3)

99 (48.5)

59 (38.1)

 

The maternal effects seen in the affected rabbits are typical of the substance-related toxicity evident in this study. The particular does showing individual adverse pregnancy outcomes are shown to be particularly severely affected, even within the group. Further, the nature of pregnancy loss – affecting the entire litter, not individual pups – is suggestive of an effect on the mother, not on the foetus. It can therefore be concluded that the test material has no direct effect on the developing organism.

Conclusions:
Under the conditions of the test, the No-Observed-Effect Level (NOEL = no difference from control) for maternal toxicity was determined to be 30 mg/kg bw/day based on clinical findings, lower gestation bodyweights, lower bodyweight gain and lower food consumption seen at 120 mg/kg bw/day and aborted pregnancies at 60 and 120 mg/kg bw/day (secondary effects to maternal toxicity). The single abortion occurring at 60 mg/kg/day is within HCD, although it is noted that the affected female showed severe effects on food consumption and reduced body weight gain or even body weight losses.
The NOEL for developmental toxicity was determined to be 60 mg/kg bw/day based on increases in post-implantation loss, increases in total and early resorption sites and lower foetal body weights at 120 mg/kg bw/day however, these effects were all considered to be secondary to maternal toxicity (low weight gain, prolonged inappetance and clinical signs).
No teratogenic effects were observed.
In conclusion, all developmental effects noted in this study are secondary and directly attributable to maternal toxicity.
Executive summary:

The developmental toxicity potential of the test material was investigated in the rabbit in a study performed in accordance with the standardised guidelines OECD 414 and US EPA OPPTS 870.3700 under GLP conditions.

The test material was administered to timed-mated New Zealand White rabbits via oral gavage in corn oil from gestation day 1 to gestation day 28 at doses of 0, 30, 60 and 120 mg/kg bw/day. Doses were selected based on a range-finding study, in which the dose-limiting toxicity was maternal inappetance; a dose group starting at 300 mg/kg bw/day was reduced to 200 mg/kg bw/day then terminated within 8 days. In the range-finding study, dosing at 200 mg/kg bw/day was therefore clearly not tolerated on the basis of maternal toxicity.

In the main study, observations of the animals included clinical signs, body weights and food consumption. Animals were sacrificed on gestation day 29. The does underwent ovarian and uterine examinations and necropsy. The foetuses were examined for external, skeletal and visceral malformations and variations.

No test material-related mortality was observed.

At the top dose, fewer does successfully completed pregnancy compared to controls; the number of viable litters at term (18) was sufficient to evaluate any possible developmental effects. In this group, one female was non-pregnant, and one female died with cause of death attributed to a dosing injury.  Neither of these can be attributed to the test material.

Consistent with the results of the dose ranging study, the principal maternal effect was a reduction in food consumption, and a clear treatment-related effect is evident on both food consumption and group mean bodyweight at the top dose level.  Individual values of animals being particularly affected within a dose group show a “clear deficit” when there is any weight loss from the previously recorded bodyweight; or a food consumption value less than half of the mean value of the top dose group.

Three animals aborted (one at 60 mg/kg bw/day and two at the top dose), and one (at the top dose) delivered prematurely.  The abortions are attributed to maternal toxicity (low weight gain, prolonged inappetance and clinical signs).  The affected animals showed significant deficits of food consumption and weight gain, even relative to the group mean values for the top dose group. These failures of pregnancy can be directly attributed to the maternal effects; maternal well-being was insufficient to maintain pregnancy.

One animal of the top dose delivered early.  The litter of 7 kits appears otherwise normal and hence no effect on development can be inferred.

Two animals (both in the top dose group) showed total litter resorptions in-utero.  These were reported as “early” resorptions.  Once again, each of these animals showed prolonged periods of markedly deficient food consumption and weight gain.  

Increased post-implantation loss was attributable to the two litters showing complete resorption, in turn a consequence of marked maternal toxicity.  When these two values are excluded from the calculation of the group mean, there is no clear increase.

The reported increases in total and early resorptions are again attributable to these two litters, both of which were reported as early resorptions.  A post-implantation loss rate of 4.6% is reported at 30 mg/kg bw/day and considered the no-effect level (i.e., no differenec from controls); the high dose rate at 6.6% is not considered biologically different.  

Lower foetal weights were significant at the high dose only. These however mirrored maternal weight change, and can be attributed to the maternal weight effect.

Under the conditions of the test, the No-Observed-Effect Level (NOEL) for maternal toxicity was determined to be 30 mg/kg bw/day based on clinical findings, lower gestation bodyweights, lower bodyweight gain and lower food consumption seen at 120 mg/kg bw/day and aborted pregnancies at 60 and 120 mg/kg bw/day (secondary effects to maternal toxicity).

The NOEL for developmental toxicity was determined to be 60 mg/kg bw/day based on increases in post-implantation loss, increases in total and early resorption sites and lower foetal body weights at 120 mg/kg bw/day however, these effects were all considered to be secondary to maternal toxicity (low weight gain, prolonged inappetance and clinical signs).

In conclusion, all developmental effects noted in this study are secondary and directly attributable to maternal toxicity.

At all dose levels tested, the test material was found not to be teratogenic in the rabbit.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 January 2014 to 31 March 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD (SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8 to 10 weeks
- Weight at study initiation: 177 to 247 g
- Housing: individually housed in solid bottom cages with nonaromatic woodchip bedding
- Diet: ad libitum
- Water: tap water available ad libitum from an automatic watering system

ENVIRONMENTAL CONDITIONS
- Temperature: 68 to 79 °F
- Humidity: 30 to 70 %
- Photoperiod: 12 hour light/dark cycle

IN-LIFE DATES: From: 21 and 22 January 2014 To: 11 February 2014
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations of the test material in the vehicle were prepared weekly and stored refrigerated at 2 to 8 °C. Formulations were prepared at nominal concentrations of 20, 60 and 200 mg/mL.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected based on information from a pilot study.
- Concentration in vehicle: 20, 60 and 200 mg/mL.
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no.: 2CK0155
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing samples were evaluated for homogeneity and concentration. 1 mL samples were collected for homogeneity during week 1 from the top, middle and bottom of the 100 and 1000 mg/kg formulations. Samples from all concentrations were taken during weeks 1 and 2 from the middle stratum for analysis of concentration. The dose formulations were analysed using HPLC/UV.

HPLC/UV CONDITIONS
Column: Agilent Poroshell 120 SB-C8 column, 3.0 x 50 mm
Gradient flow: 0.05 % trifluoroacetic acid in water (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 1.0 mL/minute.
Wavelength: 230 nm
Derivatisation: Prior to analysis, duplicate samples were derivatised in hexane and phenylmagnesium bromide then diluted with isopropyl alcohol to within the range of the calibration curve. Vehicle samples were also derivitised and then diluted using a dilution factor of 100.

RESULTS
- Homogeneity
Analysis of the low- and high-dose formulations (20 and 200 mg/mL, respectively) used for dosing the first week of study confirmed they were homogeneous as prepared as each formulation met the laboratory’s acceptance criteria of 100 ± 15 % of nominal and % RSD (Relative Standard Deviation) ≤10.
- Concentration
The test formulations used for dosing the first two weeks of study analysed between 97.8 and 101.4 % of nominal with the % RSDs ranging between 0.127 to 3.344, confirming that animals were receiving the appropriate dose levels when treated at 5 mL/kg/dose. The laboratory’s acceptance criteria for concentration were similar to that for homogeneity (100 ± 15 % of nominal and %RSD ≤10). No test material was found in the control vehicle samples.
Details on mating procedure:
- Impregnation procedure: purchased timed-mated
Duration of treatment / exposure:
Gestation day 0 to 19
Frequency of treatment:
Once daily
Duration of test:
19 days
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on information from a pilot study. In the pilot study, dose levels of 75, 150, 300, and 600 mg/kg/day were evaluated. Animals were treated orally from GD 0 to 19. No maternal or developmental toxicity was observed at these dose levels in the pilot study. Therefore, in the absence of maternal toxicity at the 600 mg/kg/day dose level in the pilot study, the high dose level in the definitive study was increased to 1000 mg/kg/day, the limit dose.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations: Morbidity, mortality, injury and availability of food and water.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily
- Examinations: Animals were removed from the cage and given a detailed clinical examination. The observations included, but were not limited to, evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, respiratory and circulatory effects, autonomic effects such as salivation, nervous system effects including tremors, convulsions, reactivity to handling and unusual behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: Gestation days 0, 3, 6, 9, 12, 15, 18 and 20. Body weight change was calculated for gestation days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-20 and 0-20. Adjusted body weight (minus gravid uterus) and adjusted bodyweight change were also calculated.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: Food consumption was measured and recorded on the corresponding body weight days and calculated for the same intervals.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20. Each surviving female was euthanised by carbon dioxide inhalation, followed by exsanguination of the abdominal vena cava and immediately subjected to a caesarean section.
- Organs examined: Dams were subjected to a complete necropsy. Special emphasis was placed on structural abnormalities or pathologic changes that may have influenced pregnancy.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: all per litter
Statistics:
All parental in-life data, gravid uterine weights, corpora lutea/dam, total implantations/dam, litter size/dam, viable foetuses/dam, total number of resorptions/dam, number of early resorptions/dam and number of late resorptions/dam were assessed using group pair-wise comparisons. Levene's test was used to assess homogeneity of group variances for each specified endpoint and for all collection intervals. If Levene's test was not significant (p≥0.01), a pooled estimate of variance (Mean Square Error) was computed from a one-way analysis of variance (ANOVA) and utilised by a Dunnett's comparison of each treatment group with the control. If the Levene's test was significant (p<0.01), then Welch's t-test was used with a Bonferroni correction for comparisons with the control group. All endpoints were analysed using two-tailed tests.
Foetal sex ratio (% males/litter), % preimplantation loss and % postimplantation loss were analysed using Arcsin-Square-Root Transformation. Data presented as percentages were transformed using the arcsin of the square root and analysed in accordance with the group pair-wise comparisons.
Pregnancy index, malformations and variations were all evaluated using Fisher’s exact test. An overall test for association between response and treatment was performed. If this test was significant (p<0.05) and there were more than two groups, then each treatment group was compared to the control group. All endpoints were analysed sing two-tailed tests. Overall testing and follow-up pair-wise testing was conducted for the control and all treatment groups that had sufficient sample size (n≥3).
Mean foetal bodyweights were analysed by covariate analysis by comparing treatment groups for each endpoint (mean of foetal bodyweights per female). The covariate was litter size. Each treatment satisfying the sample size assumption was compared to the control using Dunnett's test under the analysis of covariance model. Endpoints were analysed using two tailed tests.
Indices:
- Viable foetuses
- Postimplantation loss = ((No. implantations - No. viable foetuses) / No. implantations) x 100
- Preimplantation loss = ((No. corpora lutea - No. implantations) / No. corpora lutea) x 100
- Pregnancy index = (No. pregnant females - No. females with evidence of mating) x 100
- Foetal sex ratio
Historical control data:
A large database of historical control data for the rat was available for comparison.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Salivation was observed at least once in 1, 1 and 16 animals in the 100, 300 and 1000 mg/kg bw/day groups, respectively. Audible breathing, likely related to the salivation, was observed at least once in two, three, and ten animals in the low, mid and high dose groups respectively. Audible breathing was not heard post administration in the control group. This was considered to be a pharmacologic response to the test material in the study report however, it should be taken into consideration that the test substance is corrosive and these signs can be related to portal-of-entry effects (e.g., stomach pain and/or little substance reaching the respiratory tract).
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- Administration of the test material had no effect on maternal survival. All animals in the control group and 100 mg/kg bw/day group survived until necropsy.
- One death occurred in each of the 300 and 1000 mg/kg bw/day groups on gestation days 17 and 20, respectively. These were not attributed to test material administration.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- No effect on bodyweight or body weight change was observed in the 100 and 300 mg/kg bw/day groups.
- In the 1000 mg/kg bw/day group, mean body weights on gestation day 18 and 20 were 6 and 7 % lower, respectively, than mean control values. These differences were statistically significant and attributed to test material administration. Mean body weight gain for this group between gestation days 9 to 12 was 14 % higher than the control group and 62, 32 and 18 % lower between gestation days 12 to 15, 15 to 18 and 18 to 20, respectively. Between gestation days 0 to 20 the bodyweight gain for this group overall was 15 % lower than controls. Several animals in the 1000 mg/kg bw/day group were found to have experienced weight loss during the study. With the exception of the animal found dead on GD 20 which was found at necropsy to have a white paste-like foreign material extending the full length of the oesophagus and into the stomach (which was determined to be ingested bedding material), the cause of the weight loss for the animals in this group was attributed to lower food consumption.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- No effect on maternal food consumption was noted in the 300 and 100 mg/kg bw/day dose groups and values were comparable to mean control values.
- In the high dose group, food consumption was statistically lower than controls over GD 0 to 3 (-12 %), GD 15 to 18 (-22 %), GD 18 to 20 (-14 %) and GD 0 to 20 (-9 %). This was attributed to test material administration and correlated, particularly in late in gestation, with lower body weights and body weight gains.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- At necropsy, no effect of treatment with the test material was noted in the 100 mg/kg bw/day dose level. The few findings in this group occurred at a low incidence and were not attributed to test material administration.
- Foreign material generally identified as white in colour, which appeared to be bedding material, was observed in seven and twenty three females in the 300 and 1000 mg/kg bw/day groups, respectively. This was not observed in the control or low dose group animals. The report states that toxicological significance of this finding is unclear however, given that the test substance is corrosive, this can be considered an attenpt of animals to relieve pain in their stomach. Other macroscopic findings in the 300 mg/kg bw/day group were of low incidence and not considered to be related to test material administration.
- In the 1000 mg/kg bw/day dose group, all 25 animals were found to have white foci in the nonglandular portion of the stomach. The occurrence of this finding was considered to be test material related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
No animal on study aborted.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no treatment related effects on uterine implantation data.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
- 25, 24, 24 and 24 litters, in the control, low, mid and high dose groups respectively, produced viable foetuses to examine on GD 20.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Pregnancy indices were 100, 96, 100 and 100 % in the control, low, mid and high dose groups, respectively.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no treatment related effects on uterine implantation data. The mean number of corpora lutea in the 300 mg/kg bw/day group was statistically lower than the control group. This was considered to be spurious as ovulation occurred prior to the start of treatment. Additionally the reduction in number of corpora lutea in this group had no impact on uterine implantation data.
- Mean gravid uterine weights, adjusted GD 20 body weights and adjusted GD 0 to 20 body weight change in the 100 and 300 mg/kg bw/day groups were comparable to the mean control values. In the 1000 mg/kg bw/day group, the mean gravid uterine weight was statistically lower than the mean control value. This was attributed to lower foetal body weights. The mean adjusted GD 20 body weight and GD 0 to 20 change were also statistically lower, which correlated with lower bodyweights and bodyweight change at GD 20 in this group.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
- In the 100 and 300 mg/kg bw/day group, no effect on foetal body weight was observed and body weights were comparable to the control group. In the 1000 mg/kg bw/day group, foetal body weights were 8.6 % lower for combined sexes and 8.1 and 8.6 % lower for males and females, respectively. This was found to be statistically significant and considered a direct consequence of the maternal toxicity.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Foetal sex ratios were not affected by the administration of the test material. Mean sex ratios ranged from 51.5 to 58.3 % and were comparable to the mean control ratio of 52.0 %.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No external malformations or variations were attributed to test material administration at all dose levels tested. Two foetuses in one litter of the 300 mg/kg bw/day group were found to have oedema and a meningoencephalocele defect and entire body discolouration and protruding tongue. These observations were only noted in two foetuses from the same litter. In the absence of any findings in the 1000 mg/kg bw/day group, these were therefore not considered to be test material related.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- No skeletal malformations or variations were attributed to test material administration. In the 100 mg/kg bw/day group, five foetuses from a single litter had bent long bones of the forelimbs (humerus, radius and/or ulna) and bent scapulae. With the exception of bent scapulae, these malformations are known to have low historical incidence in the performing laboratory and in the absence of similar malformations at higher dose groups, this was considered to be incidental. The dam with the affected litter was noted to have intervals of weight loss and low food consumption during the study and was found to have adhesions within the thoracic cavity and clear fluid at necropsy. The observations in the foetuses for this dam were attributed to the poor health of the dam during the study. In the 300 mg/kg bw/day group two foetuses from separate litters were noted to have dissimilar skeletal malformations. In the 1000 mg/kg bw/day group, two foetuses from the same litter had shortened bones of the fore and hind limbs, short or misshapen exoccipital bones of the skull and misshapen scapula. These were not attributed to test material administration. No other skeletal malformations were noted in foetuses from this group.
- Skeletal developmental variations in the treated groups were found to be comparable to the control group. Their occurrence within the treated groups were generally within the historical control data range.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In the 1000 mg/kg bw/day group, the only visceral malformation observed was a folded retina in a single foetus. This was considered to be spontaneous and unrelated to test material administration.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes

Table 1: Mean gestation bodyweights, bodyweight change, food consumption and gravid uterine weights

Day / Interval

Dose Group (mg/kg bw/day)

0

100

300

1000

Bodyweight (g)

Body weight Change (g)

Food Consumption (g/animal/day)

Bodyweight (g)

Body weight Change (g)

Food Consumption (g/animal/day)

Bodyweight (g)

Body weight Change (g)

Food Consumption (g/animal/day)

Bodyweight (g)

Body weight Change (g)

Food Consumption (g/animal/day)

0 / -

213.4

-

-

211.2

-

-

212.7

-

-

212.7

-

-

3 / 0-3

228.3

14.8

12.8

229.3

18.1

13.4

233.3

20.6**

13.2

230.2

17.5

11.3*

6 / 3-6

243.9

15.6

17.3

243.5

14.2

16.6

249.1

15.8

17.0

243.8

13.6

16.6

9 / 6-9

262.0

18.1

19.2

261.0

17.5

18.1

266.4

17.3

19.2

260.8

17.0

19.1

12 / 9-12

284.1

22.1

20.2

285.1

24.1

19.6

293.3

26.9**

20.3

286.2

25.3*

19.8

15 / 12-15

305.6

21.5

21.7

303.0

17.9

21.4

309.5

16.2

22.6

294.2

8.1**

20.1

18 / 15-18

337.6

32.0

24.3

335.6

32.7

23.4

343.4

34.0

23.7

316.0*

21.7**

19.0**

20 / 18-20

370.9

33.2

21.4

364.3

28.7

20.4

377.9

34.5

22.8

345.5*

27.2*

18.4*

- / 0-20

 

Overall change: 157.4

Overall mean: 19.5

 

Overall change: 153.1

Overall mean: 18.9

 

Overall: 165.0

Overall mean: 19.7

 

Overall change: 133.3**

Overall mean: 17.7**

Adjusted Final Bodyweight (g)

20

296.8

294.9

305.7

278.6*

Adjusted weight Change from Day 0 (g)

20

83.4

83.7

92.8

66.4**

Gravid Uterine Weight (g)

20

74.1

69.4

72.2

66.9*

*p < 0.05

**p < 0.01

 

Table 2: Mean foetal bodyweights

Dose Group (mg/kg bw/day)

Mean Bodyweight of Males

(g)

Mean Bodyweight of Females

(g)

Mean Bodyweight of Males & Females

(g)

0

4.29 (4.30)

4.07 (4.08)

4.19 (4.20)

100

4.19 (4.18)

3.96 (3.94)

4.09 (4.07)

300

4.24 (4.24)

4.05 (4.05)

4.15 (4.15)

1000

3.94 (3.95)**

3.73 (3.74)**

3.83 (3.84)**

**p < 0.01

( ) Least square mean

Conclusions:
Under the conditions of the test, the NOAEL for developmental toxicity and maternal toxicity were 300 mg/kg bw/day based on lower foetal body weight and lower bodyweight, bodyweight gain and food consumption observed in dams treated with 1000 mg/kg bw/day. There were no teratogenic effects recorded in the study.
Executive summary:

The developmental toxicity potential of the test material was investigated in the rat in a study performed in accordance with the standardised guidelines OECD 414 and US EPA OPPTS 870.3700 under GLP conditions.

The test material was administered to timed-mated Crl:CD (SD) rats via oral gavage in corn oil from gestation day 0 to gestation day 19 at doses of 0, 100, 300 or 1000 mg/kg bw/day. Observations of the animals included clinical signs, body weights and food consumption. Animals were sacrificed on gestation day 20. The dams underwent ovarian and uterine examinations and necropsy. The foetuses were examined for external, skeletal and visceral malformations and variations.

No treatment-related effect was observed on survival. The death of one animal in each of the 300 and 1000 mg/kg/day dose groups was not considered test material related. Salivation and audible breathing were observed in the treated groups and considered a pharmacologic response to the test material in the report, but considered likely related to the corrosive properties of the test substance. No effect of treatment at the 100 and 300 mg/kg/day dose levels was observed on gestation body weights, body weight change, or food consumption. At the 1000 mg/kg/day dose level, maternal toxicity was observed due to statistically significant reduced body weights/body weight gains mainly during late pregnacy, and on lower food consumption. A common finding in all the 1000 mg/kg/day animals only at macroscopic examination was white foci in the nonglandular portion of the stomach. Another finding included foreign material generally identified as white in colour and having the appearance of bedding was observed in 7/25 and 23/25 females in the 300 and 1000 mg/kg/day dose groups, respectively, but not observed among the control or 100 mg/kg/day animals. The reason for these animals ingesting the bedding and its toxicological significance in relation to the test material are stated to be unclear in the report, but can be explained as an attenpt of animal to relieve pain in their stomach due to ingestion of a corrosive substance.

No effect of the test material at dose levels ≤1000 mg/kg/day was observed on pregnancy indices, uterine implantation data, foetal sex ratios, or foetal external, visceral, or skeletal examinations. Lower foetal body weights (8 % lower than controls) were observed at 1000 mg/kg/day. This effects was seen only at a dose level where maternal food consumption and bodyweight gain were impaired and is considered to be a direct consequence of the maternal toxicity.

Thus, the No-Observed-Adverse-Effect Level (NOAEL) for maternal and developmental toxicity was 300 mg/kg/day. There were no teratogenic effects, at any tested dose level in the study.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
60 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
The quality of the database is high.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In the first key study (Schroeder 2014) the developmental toxicity potential of the test material was investigated in the rabbit.

The test material was administered to timed-mated New Zealand White rabbits via oral gavage in corn oil from gestation day 1 to gestation day 28 at doses of 0, 30, 60 and 120 mg/kg bw/day. Doses were selected based on a range-finding study, in which the dose-limiting toxicity was maternal inappetance; a dose group starting at 300 mg/kg bw/day was reduced to 200 mg/kg bw/day then terminated within 8 days. In the range-finding study, dosing at 200 mg/kg bw/day was therefore clearly not tolerated on the basis of maternal toxicity.

In the main study, observations of the animals included clinical signs, body weights and food consumption. Animals were sacrificed on gestation day 29. The does underwent ovarian and uterine examinations and necropsy. The foetuses were examined for external, skeletal and visceral malformations and variations.

No test material-related mortality was observed.

At the top dose, fewer does successfully completed pregnancy compared to controls; the number of viable litters at term (18) was sufficient to evaluate any possible developmental effects. In this group, one female

was non-pregnant, and one female died with cause of death attributed to a dosing injury.  Neither of these can be attributed to the test material.

Consistent with the results of the dose ranging study, the principal maternal effect was a reduction in food consumption, and a clear treatment-related effect is evident on both food consumption and group mean bodyweight at the top dose level.  Individual values of animals being particularly affected within a dose group show a “clear deficit” when there is any weight loss from the previously recorded bodyweight; or a food consumption value less than half of the mean value of the top dose group.

Three animals aborted (one at 60 mg/kg bw/day and two at the top dose), and one (at the top dose) delivered prematurely.  The abortions are attributed to maternal toxicity (low weight gain, prolonged inappetance and clinical signs).  The affected animals showed significant deficits of food consumption and weight gain, even relative to the group mean values for the top dose group. These failures of pregnancy can be directly attributed to the maternal effects; maternal well-being was insufficient to maintain pregnancy.

One animal delivered early.  The litter of 7 kits appears otherwise normal and hence no effect on development can be inferred.

Two animals (both in the top dose group) showed total litter resorptions in-utero.  These were reported as “early” resorptions.  Once again, each of these animals showed prolonged periods of markedly deficient food consumption and weight gain.  

Increased post-implantation loss was attributable to the two litters showing complete resorption, in turn a consequence of marked maternal toxicity.  When these two values are excluded from the calculation of the group mean, there is no clear increase.

The reported increases in total and early resorptions are again attributable to these two litters, both of which were reported as early resorptions.  A post-implantation loss rate of 4.6% is reported at 30 mg/kg bw/day and considered a no-effect level; the high dose rate at 6.6% is not considered biologically different.  

Lower foetal weights were significant at the high dose only. These however mirrored maternal weight change, and can be attributed to the maternal weight effect.

Under the conditions of the test, the No-Observed-Effect Level (NOEL) for maternal toxicity was determined to be 30 mg/kg bw/day based on clinical findings, lower gestation bodyweights, lower bodyweight gain and lower food consumption seen at 120 mg/kg bw/day and aborted pregnancies at 60 and 120 mg/kg bw/day (secondary effects to maternal toxicity).

The NOEL for developmental toxicity was determined to be 60 mg/kg bw/day based on increases in post-implantation loss, increases in total and early resorption sites and lower foetal body weights at 120 mg/kg bw/day however, these effects were all considered to be secondary to maternal toxicity (low weight gain, prolonged inappetance and clinical signs).

In conclusion, all developmental effects noted in this study are secondary and directly attributable to maternal toxicity.

At all dose levels tested, the test material was found not to be teratogenic in the rabbit.

 

In the second key study (Schroeder 2014), the developmental toxicity potential of the test material was investigated in the rat. The test material was administered to timed-mated Crl:CD (SD) rats via oral gavage in corn oil from gestation day 0 to gestation day 19 at doses of 0, 100, 300 or 1000 mg/kg bw/day. Observations of the animals included clinical signs, body weights and food consumption. Animals were sacrificed on gestation day 20. The dams underwent ovarian and uterine examinations and necropsy. The foetuses were examined for external, skeletal and visceral malformations and variations.

No treatment-related effect was observed on survival. The death of one animal in each of the 300 and 1000 mg/kg/day dose groups was not considered test material related. Salivation and audible breathing were observed in the treated groups and considered a pharmacologic response to the test material in the report, but considered likely related to the corrosive properties of the test substance. No effect of treatment at the 100 and 300 mg/kg/day dose levels was observed on gestation body weights, body weight change, or food consumption. At the 1000 mg/kg/day dose level, maternal toxicity was observed due to statistically significant reduced body weights/body weight gains mainly during late pregnancy, and on lower food consumption. A common finding in all the 1000 mg/kg/day animals only at macroscopic examination was white foci in the nonglandular portion of the stomach. Another finding included foreign material generally identified as white in colour and having the appearance of bedding was observed in 7/25 and 23/25 females in the 300 and 1000 mg/kg/day dose groups, respectively, but not observed among the control or 100 mg/kg/day animals. The reason for these animals ingesting the bedding and its toxicological significance in relation to the test material are stated to be unclear in the report, but can be explained as an attempt of animal to relieve pain in their stomach due to ingestion of a corrosive substance.

No effect of the test material at dose levels ≤1000 mg/kg/day was observed on pregnancy indices, uterine implantation data, foetal sex ratios, or foetal external, visceral, or skeletal examinations. Lower foetal body weights (8 % lower than controls) were observed at 1000 mg/kg/day. This effects was seen only at a dose level where maternal food consumption and bodyweight gain were impaired and is considered to be a direct consequence of the maternal toxicity.

Thus, the No-Observed-Adverse-Effect Level (NOAEL) for maternal and developmental toxicity was 300 mg/kg/day. There were no teratogenic effects, at any tested dose level in the study.

 

In the first supporting study (Noda, 1992), treatment during days 7-17 of pregnancy at dosages up to 400 mg/kg bw/day did not cause any maternal toxicity and had no effects on the incidence of dead or resorbed foetuses, the number of living foetuses, the sex ratio and the body weight of living foetuses. A single foetus in the highest dose group had anury; however, no visceral or skeletal malformations were observed. The incidence of a minor skeletal variation (i.e. cervical ribs), observed with another organotin compound, was also not dose-dependently increased. The NOAEL for both maternal and developmental toxicity was therefore 400 mg/kg bw/day, the highest dose level tested.

 

In the second supporting study (Ema, 2001), female rats were given the test material by gastric intubation at 0, 56, 226 or 903 mg/kg on days 0–3 or 4–7 of pregnancy. Female rats were sacrificed on day 20 of pregnancy and fetuses were examined for number, abnormality, mortality and weight. The maternal body weight gain and food consumption during the administration period was significantly decreased after administration of test substance at 903 mg/kg on days 0–3 or 4–7 of pregnancy. The pregnancy rate in the treated groups was comparable to the control value, regardless of the days on which test material was given. The incidence of pre-implantation embryonic loss was not significantly affected after administration of test material on days 0–3 or 4–7. In females having implantations, the numbers of corpora lutea, implantations, and live foetuses and the incidences of pre- and post-implantation loss in the groups given test material on days 0–3 were comparable to the controls. Although a significant increase in the incidence of post-implantation loss was observed after administration of test material on days 4–7 at 56 mg/kg, this change was small and inconsistent across doses and seems unlikely to be toxicologically significant. A significant decrease in weight of female foetuses was found after administration of test material at 903 mg/kg on days 0–3 or 4–7. It could be concluded that treatment during early pregnancy is maternally toxic at 903 mg/kg.

 

In the third supporting study (Ema, 1995), higher dosages (1000, 1500 and 2000 mg/kg bw) were administered on days 7 and 8 of gestation. Five out of the 11 females treated at 1500 mg/kg bw/day and all 6 females treated at 2000 mg/kg bw/day died approximately 2 days after beginning treatment. Other signs of maternal toxicity, like body weight loss during days 7-9 and reduced body weight gain for the entire gestation period, as well as reduced adjusted weight gain, were noted in females treated at 1500 and 1000 mg/kg bw/day. Although the foetal weight in the 1500 mg/kg bw/day group was significantly decreased, no increases in the incidence of post implantation loss was recorded at either 1500 or 1000 mg/kg bw/day. Although internal and skeletal malformations were observed in a few foetuses of the treated groups, the incidence was not statistically significantly different from the controls. No NOAEL for maternal toxicity could not be determined in this study, and 1000 mg/kg bw/day was considered a LOAEL for maternal effects. Based on the reduced pup weight observed at the maternally toxic dose of 1500 mg/kg bw/day, the NOAEL for developmental effects was 1000 mg/kg bw/day.

 

A sixth, disregarded, study is also provided (Fulcher & Watson, 2013). Although the study was recently conducted in accordance with standardised guidelines and under GLP conditions, the execution of the study was flawed. It was apparent that the dosing regimen was too low to adequately assess toxicity. Furthermore, a series of dosing errors were incorrectly identified as toxicological effects rather than physical injury. Therefore, the resulting data was considered to be unreliable and unsuitable for risk and hazard assessment purposes. The study was therefore re-conducted in order to obtain data that is useful from a hazard and risk assessment perspective.

The study was performed to investigate the effects of the test material on embryonic and foetal development following repeated daily administration by gavage to the pregnant female from Day 5 to Day 19 of gestation, which included the period of organogenesis. The study was conducted in accordance with the standardised guidelines OECD 414, OPPTS 870.3700 and JMAFF 12 NohSan No 8147 under GLP conditions.

The test material was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD (SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive, at dose levels of 25, 75, and 225/150 mg/kg bw/day (following three mortalities at 225 mg/kg bw/day, the dosage was reduced to 150 mg/kg bw/day for all high dose animals regardless of their day of gestation). A further group of twenty-four time mated females received the vehicle only (Arachis Oil BP) over the same treatment period to serve as a control.

Clinical signs, body weight change and food consumptions were monitored during the study. Any animal that died or was killed prior to scheduled necropsy was subjected to a post mortem examination. All surviving females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, foetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for skeletal development and the remaining half were examined viscerally.

There were three unscheduled deaths at 225 mg/kg bw/day and an association between these deaths and treatment appears likely.

Clinical signs for animals surviving to Day 20 of gestation did not indicate any clear effect of treatment. Body weight and body weight gain was unaffected by treatment. Food consumption was also unaffected by treatment at all dose levels.

Necropsy observations for adult females at Day 20 of gestation were restricted to dark patches on the lungs for one control animal.

The number of implantations, subsequent embryofoetal survival and litter size, sex ratio and mean foetal, litter and placental weights on Day 20 of gestation were unaffected by maternal treatment at all dose levels. There was no effect of maternal treatment on morphological development of the foetuses at 25, 75 or 225/150 mg/kg bw/day.

The No Observed effect Level (NOEL) for the pregnant female was considered to be 75 mg/kg bw/day and the NOEL for the embryofoetal survival, growth, and morphological development was considered to be at least 150 mg/kg bw/day.

Justification for selection of Effect on developmental toxicity: via oral route:

Multiple studies were available to assess this endpoint. The study selected was performed in accordance with current accepted guidelines, under GLP conditions, in a relevant species and provided a suitable NOAEL in the more sensitive model.

Toxicity to reproduction: other studies

Additional information

An expert assessment of the appropriate classification for developmental toxicity of the test material under CLP, with particular respect to the results of the key rabbit study submitted to support the registration. A detailed review of individual pregnancy outcomes was performed by an expert toxicologist and were correlated with the maternal findings in order to assess the influence of maternal toxicity to pregnancy outcome. The detailed review of the available study data for developmental toxicity comparing the observed effects against the relevant classification criteria in accordance with Regulation EC 1272/2008 (CLP) (Annex II points 3.7.2.3 and 3.7.2.4) was presented as a formal report. The review is comprehensive, well documented and directly quotes and references good quality data, the report was therefore assigned a reliability score of 2.

The CLP Regulation recognizes that certain effects on pregnancy might arise as a result of maternal toxicity, and as such might not be appropriate for classification. For classification to be appropriate “effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects”. Examples cited are: “depressed foetal weight, retarded ossification, and possibly resorptions and certain malformations in some strains of certain species.”

In the key rabbit study, all treatment-related effects on pregnancy fall within these categories, with the exception of abortion. Abortion is however generally recognised as an effect that may be maternal, and is attributed by the Study Director in the study report to maternal toxicity. The author of the review agreed with this opinion.

The maternal effects seen in the affected rabbits are typical of the test material-related toxicity evident in this study. The particular does showing individual adverse pregnancy outcomes are shown to be particularly severely affected, even within the group. Further, the nature of pregnancy loss – affecting the entire litter, not individual pups – is suggestive of an effect on the mother, not on the foetus.

In the key rat study, the only treatment-related effect on development was lower foetal weight in the presence of maternal weight gain impairment. This finding is cited in the wording of the CLP Directive: “Classification is not necessarily the outcome in the case of minor developmental changes, when there is only a small reduction in .. foetal weight .. when seen in association with maternal toxicity.”

In conclusion, there is convincing evidence that all effects on pregnancy in these studies are secondary and directly attributable to maternal toxicity.

No classification for developmental toxicity is appropriate under CLP

Justification for classification or non-classification

In accordance with points 3.7.2.3 and 3.7.2.4 of Annex II of Regulation EC 1272/2008, a thorough assessment on the appropriate classification for developmental toxicity of the test material under CLP, with particular respect to the results of the key rabbit study was performed including detailed review of individual pregnancy outcomes which were correlated with maternal findings in order to assess the influence of maternal toxicity to pregnancy outcome.

Based on the weight of evidence of the available developmental toxicity data, it was concluded that there is convincing evidence that all effects on pregnancy in the available studies are secondary and directly attributable to maternal toxicity.

Therefore the test material is not classified for reproductive and developmental toxicity according to Regulation EC no.1272/2008.