Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
1983
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
2001
Deviations:
yes
Remarks:
the conduct of the study was consistent in all important aspects to the OECD 416 (2001) except that the examination of the oestrus cycle and sperm parameters are missing.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Amines, N-C10–C16-alkyltrimethylenedi-, reaction products with chloroacetic acid
Cas Number:
139734-65-9
Molecular formula:
Not applicable (UVCB substance)
IUPAC Name:
Amines, N-C10–C16-alkyltrimethylenedi-, reaction products with chloroacetic acid
Test material form:
liquid

Test animals

Species:
rat
Strain:
other: Wistar rats Crl: (WI) BR (outbred, SPF-Quality)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: P-females: 15 weeks, P-males: 7 weeks
- Weight at study initiation: P-females: 219–299g, P-males: 236–292g
- Housing: premating - in groups of max. 4; during mating - 1:1; after mating - individually; offsprong were kept with the dam until weaning
- Diet: standard pelleted diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 13-20 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: max. 3 weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after three unsuccessful weeks: no
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations prepared during weeks 1, 8, 13, 22 and 36 were analysed to check homogeneity and accuracy of preparation (all concentrations).
Analysis of the formulations revealed values for accuracy within the range of 92% and 105% of nominal. This was considered to represent an acceptable level for formulations of this type. The low and high dose formulations were considered to be sufficiently homogenous to be used in this type of study. Several measurements were considered to be outliers due to malfunctioning of the TOC apparatus, and excluded from interpretation.
Duration of treatment / exposure:
P-males: 10 weeks prior to mating up to termination.
P-females: 2 weeks prior to mating, during mating, pregnancy and lactation.
F1-males: from weaning onwards for at least 10 weeks prior to mating up to termination.
F1 females: from weaning onwards for at least 10 weeks prior to mating, during mating, pregnancy and lactation.
F2-pups were not treated.
Frequency of treatment:
daily
Details on study schedule:
- F1 parental animals not mated until at least 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation after weaning.
- Age at mating of the mated animals in the study: at least 10 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Remarks:
based on test substance as manufactured (20% aqueous solution)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Remarks:
based on test substance as manufactured (20% aqueous solution)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
based on test substance as manufactured (20% aqueous solution)
Dose / conc.:
2 mg/kg bw/day (actual dose received)
Remarks:
based on active ingredient
Dose / conc.:
6 mg/kg bw/day (actual dose received)
Remarks:
based on active ingredient
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Remarks:
based on active ingredient
No. of animals per sex per dose:
P-generation: 24 animals of each sex per group
F1-generation: 22 to 24 animals of each sex per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on an embryotoxicity and teratogencity study with the registration substance (20% solution) admininstered by oral gavage in female Wistar rats, in which 0, 40, 100, and 250 mg/kg bw/day were dosed. Females of the 250 mg/kg bw/day dose group showed clinical signs, and reduced body weight, body weight gain, food consumption and relative food consumption when compared to the control group. No effects were seen in the 40 and 100 mg/kg bw/day groups.
Positive control:
none

Examinations

Parental animals: Observations and examinations:
Clinical signs: Yes, daily

Body weight: Yes; weekly
Mated females were weighed on gestation days 0, 7, 14 and 21 and during lactation on the same days as the weighing of the litters (days 1, 4, 7, 14, 21 and 25).

Food consumption: Yes, weekly
Food consumption of mated females was measured on gestation days 0, 7, 14 and 21 and during lactation on the same days as the weighing of the litters (days 1, 4, 7, 14, 21 and 25).

Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation performed since no effect on water consumption was suspected.

Oestrous cyclicity (parental animals):
Not determined
Sperm parameters (parental animals):
Not determined
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 ] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death wasnot determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
F0-males were killed as soon as possible after confirmation of pregnancy of F0-females. F0-females were killed as soon as possible after the lactation period.
On day 4 of lactation or shortly thereafter, F1-offspring and F2-offspring eliminated for standardisation of the litter were killed. F1-offspring not selected for mating were killed as soon as possible after weaning. F1-offspring selected for mating were killed as soon as possible after weaning of the F2-offspring.

MACROSCOPIC EXAMINATION
All parental animals (P and F1) were subjected to macroscopic examination of the cranial, thoracic, abdominal tissues and organs, with special attention to the reproductive organs and abnormalities.
Samples of the following tissues and organs were fixed in 4% formaldehyde: all gross lesions, cervix, coagulation gland, epididymides, ovaries, pituitary gland, prostate, seminal vesicles, stomach, testes, uterus, vagina.
Postmortem examinations (offspring):
F1-offspring not selected for mating and F2-offspring:
Offspring found dead or killed before day 14 of lactation were sexed and externally examined if practically possible. If possible, defects or cause of death were evaluated. The stomach was examined for the presence of milk. No pups were preserved for further examination.
Offspring found dead or killed on or after day 14 of lactation were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs. Descriptions of all macroscopic abnormalities were recorded. If possible, defects or cause of death were evaluated. No pups were preserved for further examination.
Statistics:
Body weight, body weight gain, food consumption, relative food consumption, pup weight: Dunnett-Test (variables could be assumed to follow a normal distribution)
Implantation sites, living pups at 1st litter check: Steel-test (data cannot be assumed follow a normal distribution)
Macroscopic findings, fertility index, conception rate, gestation index, percentage mating, breeding data: Fisher’s Exact Test
Reproductive indices:
Percentage mating
Fertility index
Conception rate
Gestation index
Offspring viability indices:
Percentage live males at first litter check
Percentage live females at first litter check
Percentage of postnatal loss days 0-4 post partum
Percentage of breeding loss day 5 until weaning
Percentage live males at weaning
Percentage live females at weaning
Viability index
Weaning index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Rales were observed at a low incidence and on few occasions among the high dosed animals; the high dosed females showed a slightly increased incidence of hunched posture. No nodules or masses were noted.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test item-related mortality was observed at any dose level.
One female rat from the 10 mg/kg bw/day dose group died spontanously on day 40 due to delivery difficulites. 1 male and 1 female rat from the 100 mg/kg bw/day dose group were found dead. One male rat from the high dose group was killed in extremis due to bad health.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights were decreased in:
- Males (100 mg/kg bw/day) from the 5th week of treatment onwards, which showed a statistical significant decrease during post mating.
- Females (100 mg/kg bw/day) showed a statistical significant decrease during pregnancy and lactation.
Body weight gain was statistically significantly decreased in:
- Males (100 mg/kg bw/day) during the last half of the pre-mating period.
- Females (100 mg/kg bw/day) during pregnancy.
Body weight and body weight gain for males of the 10 and 30 mg/kg dose group were unaffected.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was statistically significantly decreased in:
- Females (100 mg/kg bw/day) during the pre-mating, pregnancy and lactation periods.
Relative food consumption was statistically significantly decreased in:
- Males (100 mg/kg bw/day) during weeks 1, 3 and 5 pre-mating.
- Females (100 mg/kg bw/day) during pre-mating and the first two weeks of pregnancy.
Minor statistically significant differences between controls and 10 or 30 mg/kg bw/d were considered to be of no toxicological relevance.
Details are presented in Table A6.8.2- 2
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
Endocrine 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
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
The number of implantation sites was statistically significant decreased in dams of the 100 mg/kg bw/day dose group. The number of living pups at first litter check (day 1 of lactation) was statistically significantly decreased in the 100 mg/kg bw/day dose group. However, comparison of data from this study with all data gathered at the Test Facility of other generation studies performed in the same period indicate that the average number of implantation sites and living pups at day 1 of lactation observed in the 100 mg/kg bw/day dose group falls within the normal variation as observed in the other generation studies. Moreover, comparison of all data also demonstrates that the concurrent control values observed in the current study are relatively high. Since also no toxicological effects were seen in all other developmental and reproduction parameters of the F0- and F1-generations, the lower number of implantation sites and living pups at day 1 of lactation in the F0 generation was considered non-adverse.
Total postnatal loss between days 0 and 4 post partum was statistically significantly increased and the viability index was statistically significantly decreased in litters of the 10, 30 and 100 mg/kg bw/day dose groups when compared to the control group. However, these findings were considered to be of no toxicological significance.
Total breeding loss between days 5 and 25 post partum was statistically significantly increased in litters of the 30 mg/kg dose group when compared to the control group. This finding was considered to be of no toxicological significance.
Details are presented in Tables A6.8.2-4, A6.8.2-5 and A6.8.2-6

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: decreases in body weight, body weight gain and relative food consumption in male and female high dose animals (100 mg/kg bw/day); absolute food consumption was significantly decreased in females only
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
6 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: decreases in body weight, body weight gain and relative food consumption in male and female high dose animals (20 mg a.i./kg bw/day); absolute food consumption was significantly decreased in females only
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical appearance was unaffected when treated up to 100 mg/kg bw/day. An increased incidence in piloerection was noted in animals of the 30 and 100 mg/kg bw/day dose groups during the first two weeks of treatment. A slightly increased incidence in hunched posture was noted in males of the 100 mg/kg dose group during the first two weeks of treatment. Because these findings were transient, they were not considered to be of any toxicological significance. One female of the 30 mg/kg bw/d group showed a palpable mass at the chest from week 15 onwards. This single observation was considered to be caused by chance and thus of no toxicological significance.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test item-related mortality was observed at any tested dose level.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights of males of the 100 mg/kg bw/day dose group were statistically significantly decreased during the pre-mating and mating period.
Females of the 100 mg/kg bw/day dose group showed statistically significantly decreased body weights during the first week of the pre-mating period. Because this was a transient effect, it was not considered to be of toxicological significance. Body weight gain was statistically significantly increased in females of the 10 mg/kg dose group during weeks 3–10 of the pre-mating period. Due to the absence of a dose response relationship, this finding was considered to be of no toxicological relevance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was statistically significantly decreased in males of the 100 mg/kg bw/day dose group during the pre-mating period and the relative food consumption was also slightly lower. During several weeks of the pre mating period, absolute and relative food consumption were statistically significantly decreased in females treated with 100 mg/kg bw/day.
Results are presented in Table A6.8.2- 2.

Females of the 100 mg/kg bw/day dose group showed statistically significantly decreased (relative) food consumption during the last week of pregnancy. Because the mean of means during the pregnancy period was similar as the control group, and no effects were observed during the lactation period, this finding was not considered to be of any toxicological significance. Minor statistically significant differences arising between controls and animals receiving 10 or 30 mg/kg bw/day occurred in the absence of a dose-related response, body weight changes, and/or supportive clinical signs. Therefore, they were considered to be of no toxicological relevance.
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
Endocrine 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):
Macroscopic observations at necropsy did not reveal any alterations that were considered a result of treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Reproduction parameters and breeding data were unaffected by treatment.
Results are presented in Table A6.8.2- 4.

Effect levels (P1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
6 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
20 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity (P1)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Total postnatal loss between days 0 and 4 post partum was statistically significantly increased in litters of the 10, 30 and 100 mg/kg bw/day dose groups when compared to the control group. The increase in postnatal loss in the 10 mg/kg bw/day dose group, was mainly due to three litters in which an increased loss was observed. In one of those litters all fifteen pups died spontaneously or were killed in extremis because the dam died spontaneously due to delivery difficulties. The increase in postnatal loss in the 30 mg/kg bw/day dose group, was mainly due to one litter in which all fourteen pups died on or before day 5 post partum. This was propably due to bad health of the dam (hunched posture and body weight loss). The increase in postnatal loss in the 100 mg/kg bw/day dose group, was mainly due to two litters in which an increased loss was observed. In one of those litters all nine pups died on or before day 5 post partum. This was probably due to bad health of the dam (hunched posture, piloerection, and reduced body weight). Because this statistically significant difference was due to single observations, the number of affected litters was similar for control and treatment groups, and no treatment related change was observed, this finding was considered to be of no toxicological significance.

Total breeding loss between days 5 and 25 post partum was statistically significantly increased in litters of the 30 mg/kg bw/day dose group when compared to the control group. However, this was only a light increase, no dose related response was observed, and it was mainly due to the death of three pups in one litter of which the dam showed bad health. Thus, this finding was considered to be of no toxicological significance.

The viability index was statistically significantly decreased in litters of the 10, 30, and 100 mg/kg bw/day dose groups when compared to the control group. However, because this statistically significant difference was due to single observations, the number of affected litters was similar for control and treatment groups, and not treatment related change was observed, this finding was considered to be of no toxicological significance.
The weaning index was similar for treated and control groups.
Body weight and weight changes:
no effects observed
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
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

F1-pups
Development of pups was similar for control and treated groups.
No test-item related macroscopic findings were observed upon necropsy of F1-weanlings.
Mean body weights of pups were similar for control and treated groups.

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
20 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The numbers of dead and living pups per litter at first litter check (day 1 of lactation) were similar for control and treatment groups.

Total postnatal loss between days 0 and 4 post partum was statistically significantly increased in litters of the 10 mg/kg bw/day dose group and statistically significantly decreased in litters of the 100 mg/kg bw/day dose group when compared to the control group. The total number of affected litters by postnatal loss was statistically significantly decreased in litters of the 30 mg/kg bw/day dose group. With a absence of a dose relationsship, this was considered to be caused by chance and to be of no toxicological significance.
Number of affected litters and total breeding loss between days 5 and 25 post partum was statistically significantly increased in litters of the 10 mg/kg bw/day dose group when compared to the control group. As no dose relationship was apparent, this finding was considered to be of no toxicological significance. Total breeding loss between days 5 and 25 post partum was also statistically significantly increased in litters of the 30 mg/kg bw/day dose group. However, no dose related response was observed, and it was mainly due to the death of eight and five pups in two litters. Thus, this finding was considered to be of no toxicological significance.
The viability and weaning inidices were similar for treated and control groups.
Body weight and weight changes:
no effects observed
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
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Details on results (F2)

F2-pups
Development of pups was similar for control and treated groups.
No test-item related macroscopic findings were observed upon necropsy of F2-weanlings.
Mean body weights of pups were similar for control and treated groups.

Effect levels (F2)

open allclose all
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
20 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Table A6.8.2-1: Animal assignment for mating.

 

 

Number of animals

 

 

Control

10 mg/kg /daya

30 mg/kg/daya

100 mg/kg/daya

P mating

Male

24

24

24

24

 

Female

24

24

24

24

F1mating

Male

24

23

23

22

 

Female

24

22

23

22

a: based on test substance as manufactured (20% aqueous solution)

 

Table A6.8.2-2: Observed effects on mortality and body weights during the study.

Parameter

 

 

Control

10 mg/kg/d#

30 mg/kg/d#

100 mg/kg/d#

 

 

Generation

Male

Female

Male

Female

Male

Female

Male

Female

Mortality

 

P

 

 

 

1/24

 

 

2/24

1/24

 

 

F1

 

 

 

 

1/23

2/23

4/22

2/22

Body weight

[g]

 

 

 

 

 

 

 

 

 

Pre-mating

Week 1

Week 3

Week 11

 

P

 

260

336

492

 

272

273

n.a.

 

265

344

504

 

270

270

n.a.

 

266

353

491

 

268

271

n.a.

 

267

341

461

 

264

263

n.a.

Post-mating / Pregnancy

Day 0

Day 1

Day 21

 

 

 

n.a.

494

n.a.

 

277

n.a.

450

 

n.a.

513

n.a.

 

271

n.a.

427

 

n.a.

489

n.a.

 

272

n.a.

433

 

n.a.

452*

n.a.

 

261*

n.a.

397**

Lactation

Day 1

Day 14

Day 25

 

 

 

 

324

360

331

 

 

315

355

326

 

 

316

350

318

 

 

295**

331**

303**

Pre-mating

Week 1

Week 5

Week 10

 

F1

 

117

354

494

 

104

232

284

 

109

353

496

 

96

234

299

 

112

347

478

 

99

235

289

 

102*

323*

428**

 

93*

223

279

Pregnancy

Day 0

Day 21

 

 

 

n.a.

n.a.

 

297

453

 

n.a.

n.a.

 

311

478

 

n.a.

n.a.

 

304

469

 

n.a.

n.a.

 

289

432

Body weight gain

[%]

 

 

 

 

 

 

 

 

 

Pre-mating

Week 6

Week 11

 

P

 

58

89

 

n.a.

n.a.

 

59

90

 

n.a.

n.a.

 

57

84

 

n.a.

n.a.

 

50*

73**

 

n.a.

n.a.

Pregnancy

Day 14

Day 21

 

 

 

 

27

63

 

 

25

58

 

 

25

60

 

 

21**

52**

Pre-mating

Week 6

Week 10

[%]

F1

 

235

329

 

141

177

 

263

362

 

172**

223*

 

245

332

 

154

194

 

241

313

 

154

198

Pregnancy

Day 14

Day 21

 

 

 

 

21

53

 

 

20

54

 

 

21

54

 

 

21

50

n.a.: not applicable

*: Dunnett-test statistically significantly different compared to control (p<0.05); ** (p<0.01)
#:in terms of test substance as manufactured (20% aqueous solution)

 

Table A6.8.2-3: Observed effects on food consumption during the study.

Parameter

 

 

Control

10 mg/kg/d#

30 mg/kg/d#

100 mg/kg/d#

 

 

Generation

Male

Female

Male

Female

Male

Female

Male

Female

Food consumption

[g / animal / day]

 

 

 

 

 

 

 

 

 

Pre–mating

Weeks 1/2

Weeks 2/3

 

P

 

28

28

 

22

21

 

29

30

 

21

20

 

29

30

 

21

20

 

28

28

 

19**

18**

Pregnancy

Days 0–7

Days 7–14

Days 14–21

 

 

 

 

26

27

29

 

 

24*

26

27*

 

 

24*

27

28

 

 

22**

23**

25**

Lactation

Days 1–4

Days 7–14

Days 14–21

 

 

 

 

38

59

71

 

 

33

56

67

 

 

36

56

67

 

 

31**

52**

63*

Pre–mating

Weeks 1/2

Weeks 2/3

Weeks 5/6

Weeks 10/11

[g / animal / day]

F1

 

22

28

35

36

 

18

20

25

24

 

21

27

34

35

 

17

20

26

26

 

19*

25

31

31

 

17

20

24

23

 

18*

23*

28*

27**

 

16**

17*

21*

21

Pregnancy

Days 14–21

 

 

 

 

30

 

 

31

 

 

31

 

 

27*

Relative food consumption

[g / kg bw / day]

 

 

 

 

 

 

 

 

 

Pre–mating

Weeks 1/2

Weeks 2/3

 

P

 

93

85

 

79

77

 

94

88

 

76

74

 

94

85

 

78

75

 

89*

83

 

72**

70**

Pregnancy

Days 0–7

Days 7–14

 

 

 

 

82

78

 

 

79*

76

 

 

80

79

 

 

75**

72*

Pre–mating

Weeks 1/2

Weeks 2/3

[g / kg bw / day]

F1

 

118

111

 

121

111

 

117

110

 

120

110

 

109

102

 

118

107

 

117

103

 

116

98*

Pregnancy

Days 14–21

 

 

 

 

67

 

 

64

 

 

65

 

 

63*

n.a.: not applicable
*: Dunnett-test statistically significantly different compared to control (p<0.05); ** (p<0.01)
#:based on test substance as manufactured (20% aqueous solution)

 

 

Table A6.8.2-4: Observed effects on reproductive performance of the two generation study.

 

 

 

Control

10 mg/kg/d a

30 mg/kg/d a

100 mg/kg/d a

Parameter

 

Implantations-sites

Mean

P

15.6

14.3

14.7

13.1+

 

F1

14.0

14.9

15.1

12.6

Living pups at 1. litter check

Mean

P

15.3

13.9

14.2

12.9 +

 

F1

13.8

14.1

14.9

13.0

Postnatal loss days 0-4 p.p.

Total

P

11

41##

21#

25##

 

F1

18

38##

12

4##

Breeding loss days 5–25 p.p.

Total

P

1

3

6+

5

 

F1

0

12##

14##

3

Viability index

 

P

97

87.7##

93.8#

91.6##

 

F1

94.1

87.7

96.3

98.4

+: Steel-test significant at 5% level
# /##: Fisher’s exact test significant at 5% (#) or 1% (##) level
p.p. = post partum
a:based on test substance as manufactured (20% aqueous solution)

 

Table A6.8.2-5: Comparison of number of implantation sites in the 2-generation reproductive toxicity study (report no. 285637) and historical lab data.

 

NOTOX Project 285637; start in 2000

2-generation study; NOTOX Project 291869; start in 2000

1-generation study; NOTOX Project 270618; start in 1999

 

F0

 

Control

10 mg/kg bw/day

30 mg/kg bw/day

100 mg/kg bw/day

Group 1

Group 1

Group 2

Group 3

Group 4

Mean

15.6

14.3

14.7

13.1

13.2

13.9

13.8

14.5

14.2

SD

3.2

2.8

2.6

3.6

3.7

3.0

2.9

2.6

3.3

Number of animals

24

24

24

23

20

22

21

23

23

Range

6 - 21

9 - 20

9 - 19

4 - 19

1 - 19

6 - 19

5 - 18

9 - 19

1 - 18

Number of dams with < 10 Implantation sites (IS)

1 (6 IS)

1 (9 IS)

1 (9 IS)

2 (1 x 4; 1 x 5 IS)

3 (1 x 1; 2 x 9 IS)

2 (1 x 6; 1 x 8 IS)

1 (5 IS)

1 (9 IS)

1 (1 IS)

 

F1

Mean

14.0

14.9

15.1

12.6

14.7

---

---

---

---

SD

2.9

3.2

3.3

3.5

3.9

---

---

---

---

Number of animals

22

22

22

20

20

---

---

---

---

Range

4 - 19

5 - 20

7 - 21

1 - 18

3 - 21

---

---

---

---

Number of dams with < 10 Implantation sites (IS)

1 (4 IS)

1 (5 IS)

1 (7 IS)

2 (1 x 1; 1 x 8 IS)

1 (3 IS)

---

---

---

---

Table A6.8.2-6: Comparison of number of living pups/litters at first litter check in the 2-generation reproductive toxicity study (report no. 285637) and historical lab data.

 

NOTOX Project 285637; start in 2000

2-generation study; NOTOX Project 291869; start in 2000

1-generation study; NOTOX Project 270618; start in 1999

 

F0

 

Control

10 mg/kg bw/day

30 mg/kg bw/day

100 mg/kg bw/day

Group 1

Group 1

Group 2

Group 3

Group 4

Mean

15.3

13.9

14.2

12.9

13.9

13.8

13.4

14.4

14.1

SD

3.10

2.98

2.94

3.56

2.40

2.94

2.89

2.55

3.52

Number of animals

24

24

24

23

22

22

21

23

23

Range

6 - 21

7 - 20

8 - 19

4 - 19

6 - 17

6 - 18

5 - 18

9 - 19

0 - 18

Number of dams with < 10 pups

1 (6 pups)

2 (1 x 7; 1 x 9 pups)

2 (1 x 8; 1 x 9 pups)

2 (1 x 4; 1 x 5 pups)

2 (1 x 6; 1 x 9 pups)

2 (1 x 6; 1 x 8 pups)

1 (1 x 5 pups)

1 (1 x 9 pups)

1 (1 x 0 pups)

 

F1

Mean

13.8

14.1

14.9

13.0

13.7

---

---

---

---

SD

2.81

3.77

3.23

2.08

4.22

---

---

---

---

Number of animals

22

22

22

19

20

---

---

---

---

Range

4 - 17

5 - 20

7 - 21

8 - 17

2 - 19

---

---

---

---

Number of dams with < 10 Implantation sites (IS)

1 (1 x 4 pups)

2 (2 x 5 pups)

1 (1 x 7 pups)

1 (1 x 8 pups)

3 (1 x 2; 1 x 8; 1 x 9 pups)

---

---

---

---

Applicant's summary and conclusion

Conclusions:
In a two generation study, the registration substance (20% a.i., as manufactured) was administered to groups of male and female Wistar rats at 0 (control), 10, 30 or 100 mg/kg bw/day in water, corresponding to 2, 6 and 20 mg a.i./kg bw/day, by gavage during the pre-mating and mating period and to females also during gestation and lactation.
No treatment-related mortality occurred during the study. Parental toxicity consisted of clinical signs (only P0-animals), effects on body weights and food consumption for P0- and P1-animals receiving 100 mg/kg bw/day. Reproduction and development of the pups was not affected up to the maximum dose of 100 mg/kg bw/day.
Based on the results of this study, the parental NOAEL was established at 30 mg/kg bw/day, corresponding to 6 mg a.i./kg bw/day. The reproductive and developmental NOAEL was established at 100 mg/kg bw/day, corresponding to 20 mg a.i./kg bw/day.
Executive summary:

The registration substance (20% a.i., as manufactured) was administered to groups of male and female Wistar rats at 0 (control), 10, 30 or 100 mg/kg bw/day inwater, corresponding to 2, 6 and 20 mg a.i./kg bw/day, by gavage during the pre-mating and mating period and to females also during gestation and lactation. The two generation reproduction study was carried out in compliance with OECD guideline 416 (1983) and also with the recent version OECD 416 (2001), except that the examination of oestrus cycle and sperm parameters were not conducted in the study.

Parental toxicity

Parental toxicity was assessed by observing mortality, clinical signs, body weights, food consumption, and macroscopic examination for both generations. Mortality and macroscopic examination were unaffected in parental animals P0 and P1-generation up to 100 mg/kg bw/day. At 100 mg/kg bw/day, P0-females showed a slightly increased incidence in hunched posture and P0-animals displayed rales at a low incidence and on few occasions. No treatment-related clinical signs were observed in animals of the P1-generation.

P0 - and P1-animals of the 100 mg/kg dose group showed a reduced body weight, body weight gain, food consumption, and relative food consumption during their treatment period. 

 

Parental NOAEL= 30 mg/kg bw/day, corresponding to 6 mg a.i./kg bw/day based on decreases in body weight, body weight gain and relative food consumption in male and female high dose animals (100 mg/kg bw/day). Absolute food consumption was significantly decreased in females only.

 

Reproductive toxicity

Reproductive toxicity was assessed by observing the number of implantations at birth, mating performance, fertility indices, and breeding data. Mating performance, fertility indices, and breeding data were unaffected in animals of the P0 - and P1-generations when treated up to 100 mg/kg bw/day. P0-dams of the 100 mg/kg bw/day dose group showed a reduced number of implantation sites at necropsy and living pups at day 1 of lactation. Comparison of data from this study with all data gathered at the Test Facility of other generation studies performed in the same period indicate that the average number of implantation sites and living pups at day 1 of lactation observed in the 100 mg/kg bw/day dose group falls within the normal variation as observed in the other generation studies. Moreover, comparison of all data also demonstrates that the concurrent control values observed in the current study are relatively high. Since also no toxicological effects were seen in all other developmental and reproduction parameters of the F0- and F1-generations, the lower number of implantation sites and living pups at day 1 of lactation in the F0 generation was considered non-adverse.

The number of implantations at birth was not significant in dams of the P1-generation. 

The increase in postnatal loss in the 10 mg/kg bw/day dose group was due to three litters in which an increased loss was observed. In one of those litters all fifteen pups died spontaneously or were killed in extremis because the dam died spontaneously due to delivery difficulties. The increase in postnatal loss in the 30 mg/kg bw/day dose group was mainly due to one litter in which all fourteen pups died on or before day 5 post partum. This was probably due to bad health of the dam (hunched posture and body weight loss). The increase in postnatal loss in the 100 mg/kg bw/day dose group was mainly due to two litters in which an increased loss was observed. In one of those litters all nine pups died on or before day 5 post partum. This was probably due to poor dam health (hunched posture, piloerection, and reduced body weight). These statistically significant differences were due to single observations, the number of affected litters was similar for control and treatment groups, and no true treatment related effect was observed. There was no dose response associated with these effects. As a consequence of these observations on postnatal loss, the viability index was statistically significantly decreased in litters from all dose groups. These findings are not considered to be toxicologically relevant, they do not display a dose response and as such are not taken into account for determining the reproductive NOAEL. 

Reproductive NOAEL= 100 mg/kg bw/day, corresponding to 20 mg a.i./kg bw/day. No treatment-related adverse reproductive effects were observed for any dose tested. 

Developmental toxicity

Developmental toxicity was assessed by observing clinical signs, body weights and macroscopic examination of the pups during their lactation period. Clinical signs, body weights and macroscopic examination were unaffected in F1- and F2-pups with maternal treatment up to 100 mg/kg body weight/day. 

Developmental NOAEL= 100 mg/kg bw/day, corresponding to 20 mg a.i./kg bw/day. No treatment-related macroscopic pathological effects were observed for any dose tested. 

 

In conclusion, gavage treatment of male and female Wistar rats with the registration substance (20% a.i.) at dose levels of 10, 30 or 100 mg/kg bw/day, corresponding to 2, 6 and 20 mg a.i./kg bw/day, over two generations revealed parental toxicity in animals receiving 100 mg/kg bw/day. No treatment-related mortality occurred during the study. Parental toxicity consisted of clinical signs (only P0-animals), effects on body weights and food consumption for P0- and P1-animals receiving 100 mg/kg bw/day. Reproduction and development of the pups was not affected up to the maximum dose of 100 mg/kg bw/day. 

Based on the results of this study, the parental NOAEL was established at 30 mg/kg bw/day, corresponding to 6 mg a.i./kg bw/day. The reproductive and developmental NOAEL was established at 100 mg/kg bw/day, corresponding to 20 mg a.i./kg bw/day.