Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 2016 - January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
2-tert.-butylphenol
IUPAC Name:
2-tert.-butylphenol
Constituent 2
Chemical structure
Reference substance name:
2-tert-butylphenol
EC Number:
201-807-2
EC Name:
2-tert-butylphenol
Cas Number:
88-18-6
Molecular formula:
C10H14O
IUPAC Name:
2-tert-butylphenol
Constituent 3
Reference substance name:
o.-tert.-butylphenol
IUPAC Name:
o.-tert.-butylphenol
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): 2-tert.-butylphenol
- Physical state: liquid
- Analytical purity: 99.97%
- Purity test date: 2014-05-07
- Lot/batch No.: 1419
- Expiration date of the lot/batch: 05/2015
- Stability under test conditions: stable
- Storage condition of test material: under N2 in tightly closed container at a cool, well ventilated place
- Colour: clear

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy
- Age at study initiation: females 9 weeks, (males at least 14 weeks)
- Weight at study initiation: females 196-218 g; (males 322-355g)
- Fasting period before study: no
- Housing: No more than 5 of one sex to a cage, in polysulfon cages (59.5x38x20cm), nesting material provided inside suitable bedding bags, additionally suitable nesting material was provided as necessary. Nesting material was changed at least 3 times a week. During mating period, 1 male to 1 female in clear polysulfone cages (42.5x26.6x18.5cm) with a stainless steel mesh lid and floor, each cage tray held absorbent material which was inspected and changed daily
- Diet: commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Settimo Milanese, Italy) ad libitum
- Water: drinking water via water bottles ad libitum
- Acclimation period: approximately 2 weeks before the start of the treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 15
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 2016-02-02 To: 2016-03-04

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly intervals
- Mixing appropriate amounts with: powdered rodent diet (4RF21 Mucedola s.r.l., Settimo Milanese (MI), Italy) by initial preparation of a pre-mix followed by dilution with further quantities of diet and mixing
- Storage temperature of food: not mentioned
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated by the Analytical Chemistry Department at RTC according to RTC SOPs (RTC Study No. A1763) and the test item was found to be stable at room temperature for 8 days at the concentrations of 100 and 10000 ppm. Before treatment, analysis was performed to confirm that the method was suitable and the proposed formulation procedure was acceptable. Samples of the formulations prepared during the study (Week 1 and Week 3) were also analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in RTC SOPs for diet (80-120% for concentration and CV < 10% for homogeneity). Chemical analysis was carried out by the Analytical Chemistry Department at RTC.
Details on mating procedure:
Females were paired one to one male rat in the home cage of the male and left overnight. Vaginal smears were taken daily in the morning from the day after pairing until a positive identification of mating was made. The day of mating, as judged by the presence of sperm in the vaginal smear or by the presence of a copulation plug, was considered as Day 0 of gestation (or Day 0 post coitum). Full mating records were maintained.
Duration of treatment / exposure:
days 6 to 19 post coitum
Duration of test:
up to day 20 of gestation
Doses / concentrationsopen allclose all
Dose / conc.:
200 ppm (nominal)
Dose / conc.:
1 000 ppm (nominal)
Dose / conc.:
5 000 ppm (nominal)
Dose / conc.:
17.7 mg/kg bw/day (actual dose received)
Remarks:
see Table 1 below (Any other information on materials and methods incl. tables)
Dose / conc.:
89.51 mg/kg bw/day (actual dose received)
Remarks:
see Table 1 below (Any other information on materials and methods incl. tables)
Dose / conc.:
364.29 mg/kg bw/day (actual dose received)
Remarks:
see Table 1 below (Any other information on materials and methods incl. tables)
No. of animals per sex per dose:
24 mated females
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels were in agreement with the Sponsor based on a previous study in rats (RTC Study No.: X0310EXT)
Study design of this preliminary study:
Three groups, each of 6 mated female Sprague Dawley SD rats, received the test item in the diet (Mucedola, 4RF21) at the fixed inclusion levels of 200, 1000 and 5000 ppm (in terms of test item as supplied) during the gestation period, starting from Day 6 through Day 19 post coitum. A fourth similarly constituted group received the untreated diet and acted as a control. Body weight, daily clinical signs and food consumption were recorded during the in vivo phase. All females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examination. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, uterus weight, foetal weight and sex were recorded. All foetuses were examined for external abnormalities.
Results of this preliminary study:
No clinical signs were noted in treated females and no differences of toxicological relevance were observed in bodyweight and bodyweight gain compared to controls. Food consumption was strongly reduced in the first days of administration in the high dose group compared to the control group, but an increment was detected in the following days. A moderate reduction in the absolute weight gain was detected in all treated groups, although without a dose-relation. Gravid uterus weight, litter data and macroscopic observations were not affected by treatment and no findings were detected at the external examination of foetuses. The calculated mean daily achieved dosages for the 14 days of treatment were approximately 16, 78 and 360 mg/kg/day. On the basis of the above results, the inclusion level of 5000 ppm is considered the NOAEL (No Observed Adverse Effect Level) in this study, corresponding to the achieved dosage of 360 mg/kg body weight/day of the test item.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: each animal was observed daily, for mortality all animals were checked early in the morning and again in the afternoon, at weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: during the dosing period an additional observation for signs of reaction to treatment was performed at approx. 1,5-2 h after dosing


BODY WEIGHT: Yes
- Time schedule for examinations: all animals were weighed an Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes, measured on Days 3, 6, 9, 12, 15, 18 and 20 post coitum starting Day 0 post coitum
- Food consumption for each animal determined and mean daily diet consumption calculated: Yes, as g food/animal/day per cage
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes, achieved dosage = ppm x food consumption / body weight

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 (carbon dioxide)
- Organs examined: necropsy (including examination of the external surface and orifices), changes were noted and the abnormalities preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands which were fixed in Modified Davidson’s fluid and preserved in 70% ethyl alcohol)

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
- Other: number, sex and weight of all live foetuses, number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing), gross evaluation of placentae, uteri or individual uterine horns without visible implantations were immersed in a 20 % solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation
Fetal examinations:
- External examinations: Yes: all live foetuses
- Soft tissue examinations: Yes: approx. half per litter
- Skeletal examinations: Yes: approx. half per litter
- Head examinations: Yes
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett's test (if group variances were homogeneous) or a modified t-test (if group variances were inhomogeneous).
Statistical analysis of non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.
Indices:
Pre-implantation loss was calculated as a percentage from the formula: (no. of corpora lutea - no. of implantations) x 100/no. of corpora lutea
Post-implantation loss was calculated as a percentage from the formula: (no. of implantations - no. of live young) x 100/no. of implantations
Total implantation loss was calculated as a percentage from the formula: (no. of corpora lutea - no. of live young) x 100/no. of corpora lutea
Sex ratios of the foetuses were calculated as the percentage of males per litter
All derived values (e.g., means, percentages, ratios) were first calculated within the litter and the group values derived as a mean of individual litter values. Foetal structural deviations were expressed as the percentage of affected foetuses relative to all foetuses examined per group, as well as in terms of the mean litter percentage of affected litters.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decreases in body weight were detected in the high dose group starting from Day 9 post coitum until termination. The changes were up to 8%.
At body weight gain, a statistically significant decrease was noted on Days 9 and 20 post coitum in the high dose group when compared to control. These statistically decreases were of -105 % and -31% on Days 9 and 20 post coitum, respectively. For details see Tables 3 and 4 below (Any other information on results incl. tables).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease of approximately -50% was detected on Day 9 post coitum in females of the high dose group. A trend of recovery was noted in the subsequent days but a statistically significant decrease was still noted at the end of the study when the difference was -15% compared to the control group. For details see Table 5 below (Any other information on results incl. tables).
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease in uterus weight (-15%) was observed in females of the high dose group compared to controls. For details see Table 6 below (Any other information on results incl. tables).
Gross pathological findings:
no effects observed

Maternal developmental toxicity

Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
An increased incidence in the number of early intrauterine deaths was observed in females of the high dose group compared to the control. As a consequence, the total number of viable foetuses was decreased. For details see Table 7 below (Any other information on results incl. tables).
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
Post-implantation loss was observed in females of the high dose group. For details see Table 7 below (Any other information on results incl. tables).
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
89.51 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
number of abortions
organ weights and organ / body weight ratios
pre and post implantation loss

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
In the high dose group, the total number of viable foetuses was decreased (approximately -14%). For details see Table 7 below (Any other information on results incl. tables).
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
Litter size and litter weight was decreased in the high dose group (approximately -14%, resp. -16%). For details see Table 7 below (Any other information on results incl. tables).
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Two high dose foetuses, from two different dams, showed malformations. One foetus from dam no. 151 showed fused ribs and one foetus from dam no. 191 showed multiple alterations such as unossified ischium, pubis and basisphenoid and ulna and radius misshaped. Also these foetuses were small (foetal weight < 2.7 g), with a foetal weight of 1.90 g and 1.81 g, respectively. The other changes noted were comparable between groups. For details see Table 10 below (Any other information on results incl. tables).
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
In the high dose group two foetuses of two different dams (animal nos. 183 and 191) showed the pelvic region of the kidney extremely enlarged, unilaterally and bilaterally, respectively. One of these foetuses, from dam no. 191, also showed extreme enlargement of the ureters (bilateral). These alterations were classified as malformations. Minor alterations, classified as variations or anomalies, related to kidneys pelvi or ureters were also observed in the high dose group with severity from slight to moderate. The incidence, in terms of litters affected, of these findings reached 70% for ureters enlarged compared to 13% of the controls and 39% for pelvic dilatation compared to 4% of the control. An increased incidence in displaced testes (48% in the high dose group compared to 26% of the control group, in terms of litters affected) was also observed. The incidences of these findings were above the background range, but the maternal toxicity observed probably indicates that they may be secondary to maternal stress. For details see Table 11 below (Any other information on results incl. tables).

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
89.51 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
reduction in number of live offspring
changes in litter size and weights
skeletal malformations
visceral malformations
Remarks on result:
other: Developmental effects were found only in maternal toxic dosage.

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
364.29 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects

Any other information on results incl. tables

Table 2: Fate of females - group incidence

Negative control group Low dose Medium dose High dose
0 ppm 200 ppm 1000 ppm 5000 ppm
Initial group size 24 24 24 24
Not pregnant 0 0 0 1
Total Resorption 1 0 0 0
Unilateral implantation 0 0 0 1
With live foetuses at gestation Day 20 23 24 24 23

Table 3: Body weight of females (g) with live foetuses at gestation day 20 - group mean data

Day of phase
0G 3 6D 9 12 15 18 20
Negative control (0 ppm) (n) 23 23 23 23 23 23 23 23
mean 238.54 254.60 267.42 279.21 296.00 318.63 364.90 398.39
SD 8.56 8.96 9.50 9.28 9.29 10.66 11.33 14.82
Low dose (200 ppm) (n) 24 24 24 24 24 24 24 24
mean 235.97 251.91 265.88 275.97 293.40 313.23 360.77 395.57
SD 11.94 9.57 10.22 10.94 11.73 12.49 13.46 15.70
Medium dose (1000 ppm) (n) 24 24 24 24 24 24 24 24
mean 238.64 256.19 268.20 278.69 293.49 315.21 362.03 395.84
SD 11.21 9.70 12.26 10.62 11.31 12.28 13.89 17.39
High dose (5000 ppm) (n) 23 23 23 23 23 23 23 23
mean 235.06 251.52 262.78 262.14* 284.58* 303.57* 344.40* 367.59*
SD 9.10 8.00 9.26 10.81 12.83 12.18 15.20 18.97

 G= Gestation phase;D= Dosing/gestation phase; * = mean value of group is significantly different from control at p<0.05 (Dunnett's test)

 Table 4: Body weight gain per dayO(g) of females with live foetuses at gestation day 20 - group mean data

Day of phase
3G 6D 9 12 15 18 20
Negative control (0 ppm) (n) 23 23 23 23 23 23 23
mean 5.355 4.275 3.928 5.596 7.534 15.434 16.745
SD 2.071 1.584 1.376 1.172 1.782 2.128 2.910
Low dose (200 ppm) (n) 24 24 24 24 24 24 24
mean 5.313 4.656 3.362 5.811 6.608 15.849 17.398
SD 1.717 1.204 1.741 1.467 2.425 2.632 3.396
Medium dose (1000 ppm) (n) 24 24 24 24 24 24 24
mean 5.847 4.003 3.498 4.934 7.239 15.607 16.906
SD 1.961 1.972 1.760 1.533 1.807 3.145 3.176
High dose (5000 ppm) (n) 23 23 23 23 23 23 23
mean 5.489 3.752 -0.214* 7.481 **($) 6.629 13.610 11.595*
SD 1.56 1.209 2.119 2.347 3.853 3.797 3.586

O= mean daily body weight gain over the previous period;G= Gestation phase;D= Dosing/gestation phase

 * = mean value of group is significantly different from control at p<0.05

 ** = mean value of group is significantly different from control at p<0.01

 Statistical analysis: Dunnett's test (group variances are homogeneous); $ = Modified t-test (group variances are inhomogeneous)

Table 5: Food consumptionO(g/animals/day) of females - group mean data

Day of phase
3G 6D 9 12 15 18 20
Negative control (0 ppm) (n) 9 9 9 9 9 9 9
mean 26.87 26.48 28.63 30.21 31.03 33.06 30.40
SD 3.48 5.01 5.12 4.74 4.48 3.24 6.34
Low dose (200 ppm) (n) 10 10 10 10 10 10 10
mean 25.01 26.24 26.53 28.13 27.74 30.13 31.01
SD 2.45 1.76 4.82 2.87 2.48 6.02 3.50
Medium dose (1000 ppm) (n) 10 10 10 10 10 10 10
mean 25.45 27.81 26.02 29.93 28.79 30.70 30.11
SD 3.93 6.98 5.17 9.69 4.39 4.17 5.94
High dose (5000 ppm) (n) 9 9 9 9 9 9 9
mean 23.56 24.66 13.99* 22.74**($) 24.03* 27.19**($) 25.84
SD 3.24 3.39 4.11 3.41 2.24 1.63 2.99

O= food consumed over the previous phase starting from allocation;G= Gestation phase;D= Dosing/gestation phase

 * = mean value of group is significantly different from control at p<0.05

 ** = mean value of group is significantly different from control at p<0.01

 Statistical analysis: Dunnett's test (group variances are homogeneous); $ = Modified t-test (group variances are inhomogeneous)

Table 6: Terminal body weight, uterus weight and absolute weight gain of females with live foetuses - group mean data

Terminal body weight (g) Gravid uterus weight (g) Absolute weight gain# (g)
Negative control (0 ppm) (n) 23 23 23
mean 394.97 85.18 71.25
SD 15.75 10.99 7.36
Low dose (200 ppm) (n) 24 24 24
mean 390.97 84.33 70.65
SD 16.36 8.57 13.05
Medium dose (1000 ppm) (n) 24 24 24
mean 391.28 83.85 68.79
SD 17.84 10.16 9.05
High dose (5000 ppm) (n) 23 23 23
mean 362.08* 72.17* 54.87*
SD 19.44 16.19 6.98

# = body weight at necropsy minus gravid uterine wt., minus body wt. at Day 0 of pregnancy

 * = statistically significantly different from control group value at p<0.05

Table 7: Litter data and sex ratios - group mean data

Uterine deaths Viable young % Implantation loss (%)
Corpora Lutea Implantations Early Late Total Total M F Males Pre Post Total Litter weight (g) Mean foetal weight (g)
Negative control (0 ppm) (n) 23 23 23 23 23 23 23 23 23 23 23 23 23 23
mean 16.22 16.04 0.78 0.00 0.78 15.26 7.43 7.83 47.96 1.26 4.74 5.98 56.05 3.68
SD 1.88 2.14 1.28 0.00 1.28 2.28 2.69 1.95 12.57 3.66 7.43 7.70 8.35 0.21
Low dose (200 ppm) (n) 24 24 24 24 24 24 24 24 24 24 24 24 24 24
mean 15.79 15.54 0.54 0.04 0.58 14.96 7.13 7.83 47.84 1.62 3.85 5.42 55.30 3.71
SD 1.74 1.84 0.93 0.20 0.93 2.07 1.83 2.06 10.70 3.42 6.42 6.70 6.99 0.26
Medium dose (1000 ppm) (n) 24 24 24 24 24 24 24 24 24 24 24 24 24 24
mean 15.67 15.58 0.42 0.00 0.42 15.17 6.96 8.21 45.85 0.59 2.68 3.27 55.71 3.69
SD 2.14 2.21 0.78 0.00 0.78 2.32 2.33 2.34 12.97 2.04 4.96 5.03 7.89 0.26
High dose (5000 ppm) (n) 23 23 23 23 23 23 23 23 23 23 23 23 23 23
mean 14.57 14.52 1.39 0.00 1.39 13.13 6.09 7.04 46.55 0.52 8.93 9.11 47.05* 3.61
SD 2.76 2.71 3.16 0.00 3.16 3.48 2.37 2.51 14.62 1.74 17.82 18.04 12.28 0.25

= statistically significantly different from control group value at p<0.05

Table 8: Macroscopic observations - group incidence

Females
Negative control group Low dose Medium dose High dose
0 ppm 200 ppm 1000 ppm 5000 ppm
Number in group 24 24 24 24
Uterus: abnormal content 0 0 0 1
Not pregnant 0 0 0 1
Total Resorption 1 0 0 0
Unilateral implantation 0 0 0 1
Whole animal: No abnormalities detected 23 24 24 22

Table 9: External examination of foetuses - group incidence

No. foetuses No. litters
Group Organ Cat Observation(s) Observed Affected % Observed Affected %
Negative control (0 ppm) Whole foetus No abnormalities detected 351 149 99.43 23 - -
AN Small 351 2 0.57 23 2 8.70
Low dose (200 ppm) Whole foetus No abnormalities detected 359 355 98.89 24 - -
AN Small 359 4 1.11 24 4 16.67
Medium dose (1000 ppm) Whole foetus No abnormalities detected 364 359 98.63 24 - -
AN Small 364 5 1.37 24 3 12.50
High dose (5000 ppm) Whole foetus No abnormalities detected 302 297 98.34 23 - -
AN Small 302 5 1.66 24 3 13.04

Table 10: Skleletal examination of foetuses - group incidence

No. foetuses No. dams
Group Organ Cat Observation(s) Observed Affected % Observed Affected %
Negative control (0 ppm) Forepaw(s) AN Metacarpal(s) no ossification 4th 181 27 14.92 23 9 39.13
Forepaw(s) AN Metacarpal(s) no ossification 181 1 0.55 23 1 4.35
Hindpaw(s) AN Metatarsal(s) no ossification 181 2 1.10 23 1 4.35
Hindpaw(s) VA Metatarsal(s) incomplete ossification 4th 181 1 0.55 23 1 4.35
Ribs VA Rudimentary 14th 181 12 6.63 23 7 30.43
Skull AN Temporal incomplete ossification 181 26 14.36 23 15 65.22
Skull VA Interparietal incomplete ossification 181 1 0.55 23 1 4.35
Skull VA Parietal incomplete ossification 181 1 0.55 23 1 4.35
Sternebrae AN No ossification 181 2 1.10 23 2 8.70
Sternebrae AN Asymmetrical ossification 181 13 7.18 23 9 39.13
Sternebrae AN Asymmetrical ossification 5th 181 22 12.15 23 8 34.78
Sternebrae AN No ossification 6th 181 2 1.10 23 1 4.35
Sternebrae VA No ossification 5th 181 8 4.42 23 5 21.74
Sternebrae VA Incomplete ossification 6th 181 40 22.10 23 13 56.52
Sternebrae VA Incomplete ossification 5th 181 34 18.78 23 14 60.87
Sternebrae VA Incomplete ossification 181 10 5.52 23 8 34.78
Thoracic vertebrae VA Centrum incomplete ossification 181 11 6.08 23 7 30.43
Thoracic vertebrae VA Centrum asymmetrical dumb-bell shaped 181 4 2.21 23 3 13.04
Thoracic vertebrae VA Centrum dumb-bell shaped 181 12 6.63 23 10 43.48
Low dose (200 ppm) Forepaw(s) AN Metacarptal(s) no ossification 4th 186 33 17.74 24 10 41.67
Hindpaw(s) AN Metatarsal(s) no ossification 186 2 1.08 24 1 4.17
Hindpaw(s) AN Metatarsal(s) no ossification 4th 186 1 0.54 24 1 4.17
Hindpaw(s) VA Metatarsal(s) incomplete ossification 4th 186 1 0.54 24 1 4.17
Lumbar vertebrae AN Centrum asymmetrical dumb-bell shaped 186 1 0.54 24 1 4.17
Pelvic girdle AN Pubis incomplete ossification 186 2 1.08 24 2 8.33
Ribs VA Rudimentary 14th 186 18 9.68 24 9 37.50
Ribs VA Short 14th 186 1 0.54 24 1 4.17
Sacral vertebrae AN Arch(es) incomplete ossification 186 1 0.54 24 1 4.17
Sacral vertebrae VA Centrum incomplete ossification 186 1 0.54 24 1 4.17
Skull AN Temporal incomplete ossification 186 32 17.20 24 11 45.83
Sternebrae AN No ossification 186 2 1.08 24 2 8.33
Sternebrae AN Asymmetrical ossification 5th 186 20 10.75 24 8 33.33
Sternebrae AN Asymmetrical ossification 186 16 8.60 24 6 25.00
Sternebrae AN No ossification 6th 186 3 1.61 24 3 12.50
Sternebrae VA Incomplete ossification 6th 186 35 18.82 24 12 50.00
Sternebrae VA No ossification 5th 186 7 3.76 24 4 16.67
Sternebrae VA Incomplete ossification 5th 186 31 16.67 24 15 62.50
Sternebrae VA Incomplete ossification 186 10 5.38 24 7 29.17
Thoracic vertebrae AN Centrum no ossification 186 2 1.08 24 2 8.33
Thoracic vertebrae AN Centrum bipartite 186 1 0.54 24 1 4.17
Thoracic vertebrae VA Centrum incomplete ossification 186 7 3.76 24 5 20.83
Thoracic vertebrae VA Centrum asymmetrical dumb-bell shaped 186 1 0.54 24 1 4.17
Thoracic vertebrae VA Centrum dumb-bell shaped 186 20 10.75 24 12 50.00
Medium dose (1000 ppm) Forepaw(s) AN Metacarpal(s) no ossification 4th 188 30 15.96 24 12 50.00
Forepaw(s) AN Metacarpal(s) no ossification 188 1 0.53 24 1 4.17
Hindpaw(s) AN Metatarsal(s) no ossification 188 1 0.53 24 1 4.17
Lumbar vertebrae AN Centrum dumb-bell shaped 188 1 0.53 24 1 4.17
Lumbar vertebrae VA Centrum incomplete ossification 188 1 0.53 24 1 4.17
Pelvic girdle AN Pubis incomplete ossification 188 2 1.06 24 2 8.33
Ribs AN Wavy 188 1 0.53 24 1 4.17
Ribs VA Rudimentary 14th 188 19 10.11 24 10 41.67
Sacral vertebrae AN Arch(es) incomplete ossification 188 1 0.53 24 1 4.17
Skull AN Frontal incomplete ossification 188 1 0.53 24 1 4.17
Skull AN Temporal incomplete ossification 188 36 19.15 24 17 70.83
Skull VA Interparietal incomplete ossification 188 2 1.06 24 2 8.33
Sternebrae AN Bipartite and asymm ossification 188 1 0.53 24 1 4.17
Sternebrae AN Asymmetrical ossification 188 13 6.91 24 8 33.33
Sternebrae AN No ossification 6th 188 5 2.66 24 4 16.67
Sternebrae AN No ossification 188 4 2.13 24 3 12.50
Sternebrae AN Asymmetrical ossification 5th 188 22 11.70 24 11 45.83
Sternebrae VA Incomplete ossification 6th 188 33 17.55 24 13 54.17
Sternebrae VA Incomplete ossification 188 16 8.51 24 11 45.82
Sternebrae VA No ossification 5th 188 5 2.66 24 4 16.67
Sternebrae VA Incomplete ossification 5th 188 32 17.02 24 16 66.67
Thoracic vertebrae AN Centrum bipartite 188 2 1.06 24 2 8.33
Thoracic vertebrae AN Centrum asymmetrical ossification 188 1 0.53 24 1 4.17
Thoracic vertebrae AN Centrum no ossification 188 4 2.13 24 3 12.50
Thoracic vertebrae VA Centrum dumb-bell shaped 188 6 3.19 24 5 20.83
Thoracic vertebrae VA Centrum incomplete ossification 188 8 4.26 24 6 25.00
High dose (5000 pm) Forelimb(s) AN Ulna incomplete ossification 157 1 0.64 23 1 4.35
Forelimb(s) AN Omerus incomplete ossification 157 1 0.64 23 1 4.35
Forelimb(s) AN Radius incomplete ossification 157 1 0.64 23 1 4.35
Forelimb(s) MA Ulna mishaped 157 1 0.64 23 1 4.35
Forelimb(s) MA Radius mishaped 157 1 0.64 23 1 4.35
Forepaw(s) AN Metacarpal(s) no ossification 157 1 0.64 23 1 4.35
Forepaw(s) AN Metacarpal(s) no ossification 4th 157 35 22.29 23 12 52.17
Forepaw(s) AN Metacarpal(s) incomplete ossification 157 1 0.64 23 1 4.35
Hindlimb(s) AN Femur incomlete ossification 157 1 0.64 23 1 4.35
Hindlimb(s) AN Fibula incomplete ossification 157 1 0.64 23 1 4.35
Hindlimb(s) AN Tibia incomplete ossification 157 1 0.64 23 1 4.35
Hindpaw(s) AN Metatarsal(s) incomplete ossification 157 1 0.64 23 1 4.35
Hindpaw(s) AN Metatarsal(s) no ossification 4th 157 1 0.64 23 1 4.35
Lumbar vertebrae AN Arch(es) incomplete ossification 157 1 0.64 23 1 4.35
Lumbar vertebrae AN Centrum no ossification 157 1 0.64 23 1 4.35
Lumbar vertebrae VA Centrum incomplete ossification 157 2 1.27 23 1 4.35
Pectoral girdle AN Clavicle incomplete ossification 157 1 0.64 23 1 4.35
Pectoral girdle AN Scapula incomplete ossification 157 1 0.64 23 1 4.35
Pelvic girdle MA Pubis no ossification 157 1 0.64 23 1 4.35
Pelvic girdle MA Ischium no ossification 157 1 0.64 23 1 4.35
Ribs AN Wavy 157 1 0.64 23 1 4.35
Ribs AN Incomplete ossification 157 1 0.64 23 1 4.35
Ribs MA Fused 157 1 0.64 23 1 4.35
Ribs VA Rudimentary 14th 157 24 15.29 23 11 47.83
Ribs VA Short 14th 157 1 0.64 23 1 4.35
Ribs VA 14 ribs 157 1 0.64 23 1 4.35
Sacral vertebrae AN Arch(es) incomplete ossification 157 4 2.55 23 4 17.37
Sacral vertebrae AN Arch(es) no ossification 157 1 0.64 23 1 4.35
Sacral vertebrae AN Centrum no ossification 157 1 0.64 23 1 4.35
Skull AN Mandible incomplete ossification 157 1 0.64 23 1 4.35
Skull AN Temporal incomplete ossification 157 19 12.10 23 10 43.48
Skull AN General incomplete ossification 157 1 0.64 23 1 4.35
Skull MA Basisphenoid no ossification 157 1 0.64 23 1 4.35
Skull VA Interparietal incomplete ossification 157 2 1.27 23 1 4.35
Sternebrae AN Asymmetrical ossification 5th 157 13 8.28 23 9 39.13
Sternebrae AN No ossification 157 6 3.82 23 3 13.04
Sternebrae AN No ossification 6th 157 2 1.27 23 2 8.70
Sternebrae AN Rudimentary 5th 157 2 1.27 23 2 8.70
Sternebrae AN Asymmetrical ossification 157 10 6.37 23 5 21.76
Sternebrae VA Incomplete ossification 5th 157 29 18.47 23 14 60.87
Sternebrae VA No ossification 5th 157 9 5.73 23 6 26.09
Sternebrae VA Incomplete ossification 6th 157 23 14.64 23 12 52.17
Sternebrae VA Incoomplete ossification 157 8 5.10 23 5 21.74
Thoracic vertebrae AN Arch(es) incomplete ossification 157 1 0.64 23 1 4.35
Thoracic vertebrae AN Centrum no ossification 157 4 2.55 23 2 8.70
Thoracic vertebrae AN Centrum bipartite 157 2 1.27 23 1 4.35
Thoracic vertebrae AN Haemicentrum 157 2 1.27 23 1 4.35
Thoracic vertebrae AN Centrum asymmetrical ossification 157 2 1.27 23 2 8.70
Thoracic vertebrae VA Centrum dumb-bell shaped  157 16 10.19 23 11 47.83
Thoracic vertebrae VA Centrum asymmetrical dumb-bell shaped 157 1 0.64 23 1 4.35
Thoracic vertebrae VA Centrum incomplete ossification 157 9 5.73 23 6 26.09

Table 11: Visceral examination of foetuses - group incidence

No. foetuses No. litters
Group Organ Cat Observation(s) Observed Affected % Observed Affected %
Negative control (0 ppm) Abdomen VA Haemorrhagic 170 1 0.59 23 1 4.35
Heart AN Atrium enlarged 170 13 7.65 23 8 34.78
Kidneys AN Pelvic dilatation moderate 170 1 0.59 23 1 4.35
Kidneys AN Ectopic 170 4 2.35 23 3 13.04
Testis AN Displaced 170 6 3.53 23 6 26.09
Ureter AN Kinked moderate 170 1 0.59 23 1 4.35
Ureter AN Enlarged moderate 170 1 0.59 23 1 4.35
Ureter VA Enlarged slight 170 2 1.18 23 2 8.70
Whole foetuses - No abnormalities detected 170 146 85.88 23 - -
Low dose (200 ppm) Heart AN Atrium enlarged 173 4 2.31 24 3 12.50
Kidneys AN Ectopic 173 2 1.16 24 2 8.33
Kidneys AN Pelvic dilatation moderate 173 3 1.73 24 3 12.50
Kidneys VA Pelvic dilatation slight 173 3 1.73 24 3 12.50
Testis AN Displaced 173 3 1.73 24 3 12.50
Ureter AN Enlarged moderate 173 4 2.31 24 3 12.50
Ureter VA Enlarged slight 173 5 2.89 24 4 16.67
Whole foetus AN Generalised oedema slight 173 4 2.31 24 3 12.50
Whole foetuses - No abnormalities detected 173 149 86.13 24 - -
Medium dose (1000 ppm) Great vessels VA Innominate artery short 176 2 1.14 24 2 8.33
Heart AN Atrium enlarged 176 2 1.14 24 2 8.33
Kidneys MA Pelvic dilatation extreme 176 2 1.14 24 2 8.33
Kidneys VA Pelvic dilatation slight 176 2 1.14 24 2 8.33
Testis AN Displaced 176 5 2.84 24 5 20.83
Ureter AN Enlarged moderate 176 2 1.14 24 2 8.33
Ureter MA Enlarged extreme 176 2 1.14 24 2 8.33
Ureter VA Enlarged slight 176 6 3.41 24 4 16.67
Whole foetus AN Generalised oedema slight 176 3 1.70 24 3 12.50
Whole foetuses - No abnormalities detected 176 156 88.64 24 - -
High dose (5000 ppm) Abdomen VA Haemorrhagic 145 8 5.52 23 3 13.04
Head AN Haemorrhage 145 1 0.69 23 1 4.35
Heart AN Atrium enlarged 145 5 3.45 23 4 17.39
Kidneys AN Pelvic dilatation moderate 145 3 2.07 23 3 13.04
Kidneys MA Pelvic dilatation extreme 145 2 1.38 23 2 8.70
Kidneys VA Pelvic dilatation slight 145 8 5.52 23 6 26.09
Testis AN Displaced 145 23 15.86 23 11 47.83
Thoraric cavity AN Haemorrhage 145 1 0.69 23 1 4.35
Ureter AN Enlarged moderate 145 7 4.83 23 6 26.09
Ureter MA Enlarged extreme  145 1 0.69 23 1 4.365
Ureter VA Enlarged slight  145 16 11.03 23 10 43.48
Ureter VA Kinked slight    145 1 0.69 23 1 4.35
Whole foetus AN Generalised oedema slight 145 1 0.69 23 1 4.35
Whole foetuses - No abnormalities detected 145 94 64.83 23 - -

Applicant's summary and conclusion

Conclusions:
On the basis of the results obtained in this study, the inclusion level of 1000 ppm corresponding to the achieved dosage of 89.51 mg/kg body weight/day of O-T-BUTYLPHENOL was considered to be the NOAEL (No Observed Adverse Effect Level) for maternal toxicity and for developmental toxicity. Developmental effects were found only in maternal toxic dosage.
Executive summary:

The effects of O-T-BUTYLPHENOL were investigated after oral administration, via the diet, in female rats during pregnancy and on embryo-foetal development.

Three groups, each of 24 mated female Sprague Dawley SD rats, received the test item in the diet (Mucedola, 4RF21) at the fixed inclusion levels of 200, 1000 and 5000 ppm (in terms of test item as supplied) during the gestation period, starting from Day 6 through Day 19 post coitum. A fourth similarly constituted group received the untreated diet and acted as a control.

Body weight, daily clinical signs and food consumption were recorded during the in vivo phase. All females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examination. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, uterus weight, foetal weight and sex were recorded. All foetuses were examined for external abnormalities. Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities.

The calculated mean achieved dosages for the treatment period from Day 6 to Day 19 post coitum were approximately 18, 90 and 364 mg/kg/day respectively in low, mid- and high dose groups, in terms of test item as supplied.

No animals died during the study. The number of females with live foetuses on gestation Day 20 was 23 in the control and high dose groups and 24 in the low and mid-dose groups.

No clinical signs were noted during the study and no signs of reaction to treatment were observed during the dosing period.

Statistically significant differences in the body weight were limited up to -8% during the study, between control and high dose females. On the contrary the differences (decrease) in body weight gain were more pronounced throughout the study and particularly evident on Day 9 post coitum.

A statistically significant decrease in food consumption was detected starting from Day 9 up to Day 18 post coitum in females of the high dose group. Although a trend of recovery was noted, food consumption of the high dose group was still lower than control group on Day 20 post coitum.

A statistically significant decrease in terminal body weight, uterus weight and absolute weight gain was observed in females of the high dose group compared to controls.

An increased incidence in the number of intrauterine deaths (early and total) and post and total implantation loss, expressed as percentage, was observed in females of the high dose group compared to the control. As a consequence, the total number of viable foetuses was decreased. Litter weight was also decreased.

Animals killed at termination did not show any relevant macroscopic changes.

No abnormalities were detected at the external examination of foetuses. A total of 16 small foetuses were detected; two out of 351 in the control group, four out of 359 in low dose group, five out of 364 in the mid-dose group and five out of 302 in the high dose group.

One small foetus of the high dose group showed multiple malformations at skeletal examination: unossified ischium, pubis and basisphenoid and ulna and radius misshaped. An additional small foetus in the same group showed fused ribs.

At visceral examination, 2 foetuses of the high dose group showed major alterations localised to the kidneys and ureters. Moreover, an increase incidence (in terms of litters affected) in minor alterations, classified as variations or anomalies, always related to kidneys or ureters, was also observed. The incidences of these findings were above the background range, but the maternal toxicity observed probably indicates that they may be secondary to maternal stress.

Taking into account that some alterations in the urinary system were also noted in the control group, the findings noted at the mid-dose level could be considered incidental.

On the basis of the results obtained in this study, the inclusion level of 1000 ppm corresponding to the achieved dosage of 89.51 mg/kg body weight/day of O-T-BUTYLPHENOL was considered to be the NOAEL for maternal toxicity and for developmental toxicity. Developmental effects were found only in maternal toxic dosage.