Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
developmental 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

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)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with fatty acids, C18-unsatd., dimers
EC Number:
500-180-5
EC Name:
4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with fatty acids, C18-unsatd., dimers
Cas Number:
67989-52-0
Molecular formula:
(C15H16O2.C3H5ClO.Unspecified)x
IUPAC Name:
2-(chloromethyl)oxirane; 4-[2-(4-hydroxyphenyl)propan-2-yl]phenol
Test material form:
liquid: viscous
Details on test material:
As per IUCLID Sections 1.1. 1.2. and 4.1.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
1 Environment
Environmental conditions are maintained within the following target parameters:
Target temperature: 22 ± 3 °C
Target humidity: 30 to 70%
Lighting: Twelve hours of continuous artificial light in each twenty-four hour period
Ventilation: At least fifteen air changes per hour
Animals are housed in accordance with the 'Code of Practice for the Housing and Care of Animals Used in Scientific Procedures' and in alignment with the United Kingdom Home Office recommendations and requirements.

2 Housing
Time-mated females are individually housed in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). Certificates of analysis of bedding are provided by the supplier. The bedding is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

3 Diet and Water
The animals will be provided with Rodent 20 18C Teklad Global Pelleted Certified Diet (Envigo RMS (UK) Ltd., Oxon, UK) and tap water ad libitum. Drinking water is routinely analyzed (at least annually) and certificates of analysis for diet
are provided by the supplier. The diet and water are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

4 Environmental Enrichment
The animals are provided with environmental enrichment items: wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) or suitable alternatives. Any alternatives used are documented in the raw data for the study. Certificates of analysis for the environmental enrichment items will be provided by the supplier. These enrichment items are considered not to contain any contaminants

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
1 Administration
Time-mated females are dosed once daily by gavage, from Day 5 (post-implantation) to Day 19 of gestation (the day prior to necropsy). Where it has been demonstrated that the test item has no adverse effect on pre-implantation (in a preliminary pre-natal study or from other toxicity data such as an OECD 421 or OECD 422 study), females will be dosed once daily by gavage, from Day 3 (pre-implantation) to Day 19 of gestation. The volume of test and control item administered to each animal will be based on the most recent scheduled body weight and adjusted accordingly.

2 Dose Groups
Four dose groups (control, low, intermediate and high) each comprising 24 time-mated females (expected to achieve 20 pregnant females per group) are used. Dose levels are based on available toxicity data. The dose levels used in the study are documented as an amendment to study plan together with the vehicle and dose volume. Control animals are treated with the vehicle alone over the same treatment period and administered at the same dose volume as that oftest animals.


Group Dose Dose Concentration Group Size Animal Numbers Duration of
Level Volume (mg/mL) & Sex Treatment
(mg/kg (mLlkg)
bw/day) Females

Control 0 4 0 24 female 1-24 17 Days
Low 100 4 25 24 female 25-48 17 Days
Intermediate 300 4 75 24 female 49-72 17 Days
High 1000 4 250 24 female 73-96 17 Days

... Control animals treated with vehicle alone (Polyethylene Glycol 400)
Analytical verification of doses or concentrations:
no
Details on mating procedure:
Females are time-mated with sexually mature proven males of the same strain by the animal supplier. The day of observation of positive evidence of mating is designated Day 0 of gestation. The animal supplier identifies mated females and provides identification details of their respective mating partners.
Duration of treatment / exposure:
Day 5 (post-implantation) to Day 19 of gestation (the day prior to necropsy)
Frequency of treatment:
Once daily
Duration of test:
21 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 females per dose level
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
Full external and internal macroscopic examination of adult females. Samples of adult tissues showing macroscopic abnormalities are preserved in buffered 10% formalin for possible future histopathological examination. Any unprocessed preserved tissues will be discarded following issue of the final report.
Ovaries and uterine content:
The uterus of each adult female are examined and where possible the following recorded;
i. Pregnancy status
ii. Number of corpora lutea
iii. Gravid uterus weight
iv. Number, status (Live fetus, dead fetus, late intra-uterine death or early intra-uterine death) and intra-uterine position of implantations

For decedent females prior to Day 20 of gestation the gravid uterus weight is not be recorded. The ability to assess the individual status for implantations from decedent animal will be dependent on the stage of gestation.
Fetal examinations:
For fetuses from decedent females prior to Day 20 of gestation, examination of the fetuses will normally be restricted to external appearance (if age permits this evaluation). Each individual fetus from all litters at Day 20 of gestation will be examined and the following will be recorded:
i. External fetal findings
ii. Fetal weight
iii. Fetal sex
iv. Placental weight
v. Skeletal and visceral fetal findings
After external examination, approximately half of each litter will be fixed in 70% Industrial Methylated Spirit (IMS), processed for skeletal evaluation and finally stored in 50% glycerol. The remainder of each litter will be fixed in bouins fluid for visceral evaluation by microdissection and finally stored in 10% neutral buffered formalin.
Statistics:
Where considered appropriate, quantitative data will be subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance will be achieved at a level ofp<0.05. Typically analysis will be performed on, but not necessarily restricted to, the following parameters:
Body weight and body weight change (including adjustment for the contribution of the gravid uterus), food consumption, gravid uterus weight, litter data, fetal litter and placental weights and fetal evaluation parameters (including external, skeletal and visceral findings).
Normally, the data are first assessed using Shapiro-Wilk test for normality and Bartlett's test for homogeneity of variance. If the data are considered to be normal and homogeneous, parametric assessment, one way analysis of variance and, if significant, Dunnett's multiple comparison test will be employed. Where the data are considered not to be normal or non homogeneous, non-parametric assessment, Kruskal-Wallis and, if significant, pairwise analysis of control values against treated values using the Mann-Whitney 'U' test will be used.
Flags for significance between control and treated groups (* for p<0.05, ** for p<0.01 and, where appropriate, *** for p<0.001) will be indicated in the tables.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Description (incidence and severity):
A summary incidence of daily clinical observations is given in Table 2. Individual data are presented in Appendix 1.

Females treated with 1000 mg/kg bw/day showed episodes of increased salivation between Days 4 and 19. Isolated incidences of noisy respiration were also evident in three females from this treatment group on Days 7, 15 or 16. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and in isolation are considered not to be of toxicological significance.

No toxicologically significant clinical signs were evident in females treated with 300 or 100 mg/kg bw/day.

One female treated with 300 mg/kg bw/day had noisy respiration on Day 19 only, a further female treated with 300 mg/kg bw/day had generalized fur loss between Days 17 and 20 and one female treated with 100 mg/kg bw/day had increased salivation on Day 3 only. In isolation, these were considered to be incidental and unrelated to the test item.
Description (incidence):
One female (no. 63) treated with 300 mg/kg bw/day was found dead on Day 20 of gestation. No clinical signs of toxicity were evident prior to death. One fetus was observed to have been born dead and at necropsy, six dead fetuses were present in the left horn of the uterus and seven dead fetuses were present in the right horn of the uterus. In the absence of any similar effects evident in the remaining females, this death was considered to be incidental and unrelated to treatment.

There were no further unscheduled deaths.
Description (incidence and severity):
Group mean body weights and standard deviations are given in Table 3 and presented graphically in Figure 1. Group mean body weight gains and adjusted body weights and standard deviations are given in Table 4 and Table 5. Individual data are given in Appendix 2, Appendix 3 and Appendix 4.

Body weight and body weight gain, including adjustment for the contribution of the gravid uterus, was unaffected by treatment at 100, 300 or 1000 mg/kg bw/day.

Statistical analysis of the data did not reveal any significant intergroup differences.
Description (incidence and severity):
Group mean food consumptions are given in Table 6 and presented graphically in Figure 2. Individual data are given in Appendix 5.

Food consumption during gestation was unaffected by treatment at 100, 300 or 1000 mg/kg bw/day.

Statistical analysis of the data did not reveal any significant intergroup differences.
Description (incidence and severity):
Daily visual inspection of water bottles did not reveal any overt intergroup differences.
Description (incidence and severity):
A summary incidence of female necropsy findings is given in Table 7. Individual data are given in Appendix 6.

No treatment-related abnormalities were detected during the macroscopic examination of the surviving pregnant females at termination on gestation Day 20.

One female (no. 65) treated with 300 mg/kg bw/day had brown staining around the anus and gaseous distension in the stomach and gastro-intestinal tract. A further female (no. 49) from this treatment group had generalised fur loss. In isolation, these were considered incidental and unrelated to treatment.

Maternal developmental toxicity

Description (incidence and severity):
Females treated with 1000 and 300 mg/kg bw/day showed a statistically significant increase in the number of female fetuses (7.4 and 7.1, respectively). Group mean values were within historical control ranges (0-12 [group mean ±S.D.: 6 ±2], refer to Annex 5) and a true dose-related response was not evident. The number of male fetuses was also comparable to controls and therefore the intergroup differences were considered not to be of toxicological importance

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: A true dose related response was not evident, therefore, the intergroup differences were considered not to be of toxicological significance.

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Summary fetal external findings, visceral findings, skeletal findings and skeletal development are given in Table 9 to Table 11. Individual data are given in Appendix 9 to Appendix 11.

Neither the type, incidence nor the distribution of findings observed during external examination of the fetuses at necropsy on gestation Day 20 and subsequent detailed visceral and skeletal examination indicated any adverse effect of maternal exposure on fetal development.

A statistically significant increase in the number of fetuses/litters showing a kinked ureter was evident at all treatment groups (10.9%, 9.1%, 8.5% for 100, 300, 1000 mg/kg bw/day groups, respectively). The group mean control value (2.8%) was below the historical control range (3.9 - 20% [group mean ±S.D.: 14.1% ±4.8], refer to Annex 7), whereas group mean values for all treated groups were within historical control range. A true dose related response was also not evident, therefore, the intergroup differences were considered not to be of toxicological significance.

A statistically significant reduction in the number of fetuses/litters (17.1%) showing ossification of the ventral arch of vertebra 1 was evident at 1000 mg/kg bw/day. Although the group mean value was slightly below the historical control range (19.5-43.3% [group mean ±S.D.: 32.0% ±6.4], refer to Annex 7) , the observation of one variant at a lower incidence compared with controls is not significant when evaluated in isolation. In the absence of any particular pattern of abnormal skeletal development of skeletal structures affecting treated fetuses, the observation of one affected skeletal structure can be considered unlikely to represent true developmental abnormality.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: A true dose related response was not evident, therefore, the intergroup differences were considered not to be of toxicological significance.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The oral administration of 4,4'-lsopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with fatty acids, C18-unsatd., dimers ERRCF #62 to pregnant rats by oral gavage from gestation Days 3 to 19 at dose levels of 100, 300 or 1000 mg/kg bw/day did not result in any treatment-related effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for the pregnant female was considered to be greater than 1000 mg/kg bw/day.

No treatment-related adverse changes were detected in the offspring parameters measured or on embryofetal development. The NOAEL for developmental toxicity was therefore considered to be greater than 1000 mg/kg bw/day.
Executive summary:

Introduction

The study was performed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis.

The study was designed to comply with the following guidelines:

• US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)

• Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)

• OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)

• Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Methods

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 3 and 19 of gestation inclusive at dose levels 100, 300, and 1000 mg/kg bw/day, employing a dose volume of 4 mL/kg bw. A further group of twenty-four time mated females was exposed to the vehicle only (Polyethylene Glycol 400) to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study.

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

Results

Test Item Analysis

Samples were taken of each test item formulation. The results indicate that the prepared formulations were within ±5% of the nominal concentration, thereby confirming the acceptability for the dose preparations.

Mortality

One female treated with 300 mg/kg bw/day was found dead on Day 20 of gestation. One fetus was observed to have been born dead and at necropsy, six dead fetuses were present in the left horn of the uterus and seven dead fetuses were present in the right horn of the uterus. In the absence of any similar effects evident in the remaining females, this death was considered to be incidental and unrelated to treatment.

There were no further unscheduled deaths.

Clinical Observations

Females treated with 1000 mg/kg bw/day showed episodes of increased salivation during the treatment period. Isolated incidences of noisy respiration were also evident in three females from this treatment group. No toxicologically significant clinical signs were evident in females treated with 300 or 100 mg/kg bw/day.

Body Weight

No adverse effect was detected in body weight development.

Food Consumption

No adverse effect was detected on food consumption.

Water Consumption

No adverse effect on water consumption was detected.

Post Mortem Studies

No macroscopic abnormalities were detected in the surviving females.

Litter Data and Litter Placental and Fetal Weights

No treatment-related effects were detected in the uterine parameters examined, in fetal viability or in growth and development.

Fetal Examination

No treatment-related effects were detected in fetal external findings. No treatment-related adverse effects on skeletal development were detected or in the type and incidence of visceral findings in fetuses from females treated with 100, 300 or 1000 mg/kg bw/day.

Conclusion

The oral administration of 4,4'-lsopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with fatty acids, C18-unsatd., dimers ERRCF #62 to pregnant rats by oral gavage from gestation Days 3 to 19 at dose levels of 100, 300 or 1000 mg/kg bw/day did not result in any treatment-related effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for the pregnant female was considered to be greater than 1000 mg/kg bw/day.

No treatment-related adverse changes were detected in the offspring parameters measured or on embryofetal development. The ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity was therefore considered to be greater than 1000 mg/kg bw/day.