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EC number: 201-807-2 | CAS number: 88-18-6
Developmental toxicity / teratogenicity
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:
- 2 017
- 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
- Reference substance name:
- 2-tert.-butylphenol
- IUPAC Name:
- 2-tert.-butylphenol
- 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
- 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
Constituent 1
Constituent 2
Constituent 3
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.
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