Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Oct - 28 Nov 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Adopted in 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,4,6-trimethylphenol
EC Number:
208-419-2
EC Name:
2,4,6-trimethylphenol
Cas Number:
527-60-6
Molecular formula:
C9H12O
IUPAC Name:
2,4,6-trimethylphenol

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: The Lab Animals Breeding Center “Pushchino”, Branch of Institute of Bioorganic Chemistry RAS:Puschino, Moscow region, Russia (www.spf-animals.ru)
- Females: Nulliparous and non-pregnant
- Age: Approximately 12 weeks at the initiation of dose administration on post-mating day 5
- Weight at study initiation: 184 g ± 15 g, N = 98 g
- Housing: Solid bottom polycarbonate cages (Type IV, 598 х 380 х 200 mm (LxWxH), 1820 cm sq., Tecniplast s.p.a.) with bedding. Females were group housed until mating, cohabited with males during mating (M/F ratio: 1:1), pregnant females were individually housed until scheduled sacrifice on gestation Day 20
- Diet: Mmucedola 4RF21 (Mmucedola s.r.l., Milanese MI, Italy) ad libitum
- Water: Filtered tap water ad libitum
- Acclimation period: Approximately 9 weeks

DETAILS OF FOOD AND WATER QUALITY
No known contaminants in the feed or water at levels that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES
From: 12 Oct 2021 To: 28 Nov 2021

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item formulations were prepared every four days as a suspension of the test item in corn oil. The required amount of the test item (weighed separately for each concentration) was mixed with a calculated volume of corn oil and stirred using a magnetic stirrer until homogenous. The total volume of each concentration was aliquoted to the required volumes of days of administration and stored in tightly closed glass jars at room temperature. For the control group, the required aliquots of corn oil were placed in labelled jars and stored at room temperature. A daily record of the usage of formulation was maintained based on weights.

VEHICLE
- Justification for use and choice of vehicle: Test item has limited solubility in water
- Concentration in vehicle: 5, 20, 80 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. : MKCN9742
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test item in the vehicle (corn oil) prepared at concentrations of 5 and 80
mg/mL was confirmed following storage for 4 days at room temperature (the temperature
range, 20 – 25 °C) during the method validation study (Study no. 797/21). Analysis of formulations for homogeneity and concentration during the dosing period was conducted in the test facility at the beginning, in the middle, and at the end of the in-life phase using a validated method.
For homogeneity analysis, quadruplicate samples were collected from the top, middle, and
bottom strata of each dosing formulation prepared during the study. For concentration
analysis, quadruplicate samples were collected from the middle stratum of each dosing
formulation (including the vehicle control group) prepared during the study. Samples
collected from the mean stratum for homogeneity analysis were used for this purpose.
Acceptance criteria for the formulations analysis are based on the test item in vehicle
composition as a suspension. The actual concentration of analyzed samples collected from
the mean stratum of formulations should be within the range of 85% - 115% of the target
concentration. The acceptance criteria for homogeneity are RSD<15% (for suspensions),
with the mean concentration within 85 -115% of the target concentration.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1:2
- Length of cohabitation: Until mating. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage
- Further matings after two unsuccessful attempts: not specified
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 of pregnancy
Duration of treatment / exposure:
Day 5 to 19 of gestation
Frequency of treatment:
Daily
Duration of test:
From start of mating to last necropsy on Day 20 of gestation
Doses / concentrationsopen allclose all
Dose / conc.:
25 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
400 mg/kg bw/day
No. of animals per sex per dose:
24 to 25 females with confirmed mating in control and low dose group. 25 females with confirmed mating in mid and high dose group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosage levels were selected for the study based on available data from a previously conducted dose range finding study (796/21 of BTC/BTL IBCh RAS). No mortality and morbidity were observed in female SD rats on the test item (with a purity of 82 %) at the doses of 100 and 400 mg/kg bw/day during 14-day oral treatment. The 400 mg/kg bw/day dose caused in 1/4 female rats transient hypotonia and ataxia, inability to move with a temporary prone position, diarrhoea, and salivation. A dose-related increase in the mean liver and kidney absolute and relative weights were revealed.
- Rationale for animal assignment: Before the beginning of the treatment period, the females with confirmed mating were allocated to the experimental groups according to a stratification procedure so that the average body weight of each group did not statistically differ. Females with the same day of gestation were allocated to a different group.
- Time of day for (rat) dam blood sampling: At scheduled necropsy on Day 20 of gestation (10:00 - 13:00 hours). Animals were not fasted prior to blood collection.


Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once in the morning and once in the afternoon at the same time, for morbidity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each female was observed for signs of toxicity approximately 15 - 60 min following dose administration. In addition, the presence of findings at the time of dose administration was recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Gestation Day 0, 5, 8,11,14,17, and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: A complete necropsy was conducted on all females at scheduled termination. Necropsy
included examination of the external surface of the body, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal and pelvic cavities including viscera.
- Sacrifice on Gestation Day 20 of gestation
- Organs examined: Thyroid gland, Uterus and ovaries
- Organs weighed: Liver, kidney, thyroid and brain
- Histopathology: Thyroid gland

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
Blood sampling:
- Plasma: No
- Serum: Yes
- Volume collected: not indicated
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: Yes
Statistics:
Continuous data variables (mean body weights and food consumption data) were analyzed by multi-factor analysis of variance (ANOVA-2), followed by the Duncan test, to determine inter-group differences. Former implantation sites, number of corpora lutea, implantation loss indices, hormones concentration values, organ weights were analyzed by parametric one-way analysis of variance (ANOVA). If the results of the ANOVA were significant (0.05), Dunnett's test is applied to the data to compare the treated groups to the control group. The t-test was used additionally to compare each dose group with the control value. The number of male and female fetuses per litter, AGD value, and mean value of affected fetuses per litter were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine the intergroup difference, and the t-test was applied to the AGD value to compare each dose from the control value. The fetal body weight was analyzed by sex as well as for both sexes combined using a one-way analysis of variance (ANOVA) as described above, and the t-test was applied to compare each dose from the control value. Additionally, statistical analysis for fetal body weight was done using covariant analysis with litter size as a covariant.
Indices:
Females with total resorptions (N)
Females with all dead fetuses (N)
Females with live fetuses (N)
Corpora lutea (N per animal)
Implantation sites (N per animal)
Pre-implantation loss (N per animal, % of implantation sites):(Number of corpora lutea – Number of -implantation sites) / Number of corpora lutea
Fetuses (N per animal)
Live fetuses (N per animal, % of implantation sites)
Dead fetuses (N per animal, % of implantation sites)
Resorptions + Scars (N per animal, % of implantation sites)
Implantation scars (N per animal, % of implantation sites)
Resorptions – early (N per animal, % of implantation sites)
Resorptions – late (N per animal, % of implantation sites)
Post-implantation loss (N per animal, % of implantation sites): (Number of implantation sites – Number of live fetuses) / Number of implantation sites
Historical control data:
Historical control data are shown in appended Tables

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The test item related external observations were noted in the 400 mg/kg bw/day dose group. Prone position was recorded for one female (No.70) following GD 13 - 15 dose administration. This clinical observation was temporary, observed approximately 30 - 40 min after dosing and was associated with hypersalivation on GD 13. The observation was considered adverse.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There was no morbidity and mortality of females caused by the test item administration.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Summary data of body weight and body weight gain for pregnant females are presented in Table 1 and 2 under 'Any other information an results incl. tables'.
A slight decrease in the mean body weight of pregnant females at 400 mg/kg bw/day (4.6 % compared to the control group) towards the end of the dosing period was not significant. However, body weight gain was statistically reduced in the high dose group starting from GD 17 compared to the control (6.5%). By GD 20, the body weight gain in the high dose group was decreased by 8.3 % compared to the control group.
However, the mean gravid uterus weight in the 400 mg/kg bw/day dose group was less than the control (17.4 % (p < 0.05)) and the maternal body weight without gravid uterus did not statistically differ from the body weight of control females. Therefore the treatment-related bodyweight effects were not considered to be adverse.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean food consumption in the 25, 100, and 400 mg/kg bw/day treatment groups was similar to that of the vehicle control group during all study days.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Females at 400 mg/kg bw/day had slightly increased relative kidney (+4.3%) and thyroid (+6.8%) weights. However, this change was not significant compared to the control group and were not considered to be biologically relevant.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No gross findings related to the test item administration were observed.

Spleen deformation was found in one female from the 100 mg/kg bw/day dose group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No histological alterations in the thyroid gland, which are considered to be associated with the test item treatment, were observed. The incidence of follicular cell hypertrophy was approximately the same among all groups (7/23, 7/24, 5/24, 5/23 in control, low, mid and high dose groups respectively). This finding was of slight or moderate grade in single females, including one control, not correlated to the change in thyroid weight and thyroid hormone levels, and so considered not test item related.
The incidence of ultimobranchial cyst was approximately the same among all groups (7/23, 7/24, 11/24, 9/23 in control, low, mid and high dose groups respectively). This finding was considered congenital, not treatment-related.

Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
There were no significant changes in the T3, T4, and TSH hormone levels in treated females compared to the control vehicle group.
The mean level of TSH in the 25 mg/kg bw dose group was lower compared to values in other groups; however, this change is considered incidental, not test item related. Considering that the level of T4 was unaffected, it was assumed that the TSH level did not change under the test item influence. There were no indications of TSH-mediated thyroid gland activation, such as a significant increase in thyroids weight.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
The mean number of corpora lutea was approximately the same among groups. However, the mean number of implantation sites was decreased in the 400 mg/kg bw/day dose group (by 17.8% compared to control vehicle group, p < 0.05). Accordingly, the mean number of pre-implantation loss and percentage of pre-implantation loss per group were increased: 3.2 ± 3.4 and 1.5 ± 1.7 (p < 0.05) in animals of the high dose and control group, respectively and 26.8% and 12.1% (p < 0.01) in animals of the high dose and control group, respectively. The percentage of pre-implantation loss in the 400 mg/kg bw/day dose group was also statistically significantly higher than the historical control value of 16.7%. According to previous studies, implantation sites in rats can be visualized (using intravenously Evans blue) 5 days 12 h later mating [Novaro V. et al., 2002; Hamilton G. et al. 1994]. In this study, the test item administration started at this time (approximately 5.5 days after mating). Therefore, it is likely that the first administered dose of 400 mg/kg bw/day can adversely affect the implantation process.
Summary data are shown in Table 3 under 'Any other information an results incl. tables'.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no total litter losses by resorption.
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
The mean number of fetuses per animal was reduced in the 400 mg/kg bw/day dose groups (by 18%, p < 0.05, compared to the control group). This change correlated to the decrease in the number of implantation sites in this group discussed above and assumed to be test item related.
The percentage and the mean number of early and late resorptions and total post-implantation loss were not statistically significantly changed compared to the control vehicle group. The number of females with post-implantation loss was approximately the same in all groups (13/23, 12/24, 8/24, 11/23 in control, low, mid and high dose group respectively. Thus, in the 400 mg/kg bw/day dose group, the decrease in the mean number of fetuses per uterus was associated with the pre-implantation loss. No increase in post-implantation loss was observed for the test item at doses of 25, 100 and 400 mg/kg bw/day.
Summary data are shown in Table 4 under 'Any other information an results incl. tables'.
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead foetuses were observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
All pregnant females reached GD 20
Changes in number of pregnant:
no effects observed
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
behaviour (functional findings)
Remarks on result:
other: One female in the 400 mg/kg bw/day dose group was prone following dosing on GD 13 - 15

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight of male fetuses was slightly decreased (by 5.2 %) for doses of 100 and 400 mg/kg bw/day compared to the control vehicle group. This change correlated to the increased incidence of external observation of “small fetus” in these groups and was considered test item-related. However, the decrease in fetal body weight was not statistically significant and therefore considered non-adverse.
The test item treatment did not change the body weight of female fetuses and mean values for all fetuses.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
All fetuses were found to be alive.
Changes in sex ratio:
effects observed, treatment-related
Description (incidence and severity):
The mean number of males per litter was statistically significantly reduced in the high dose group compared to the control vehicle group (p < 0.05). The percentage of males was decreased in this group (44.7%) compared to the value in the control group (51.9%). A decreased percentage of males per group was observed for the 25 and 100 mg/kg bw/day dose groups (46.4% and 46.9% respectively). The reduction in ratio of male and female fetuses was not statistically significant, without clear dose-response relationship and was within historical control range (53.0% for 70 litters; min–max per litter, 14–79%) and considered to be non-adverse.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
The mean number of males per litter in the 400 mg/kg bw/day dose group was statistically reduced compared to the control (p < 0.05), and the percentage of males
was decreased in this group (44.7%) compared to the value in the control group (51.9%). A decrease in the percentage of males per group was observed at 25 and
100 mg/kg bw/day dose level (46.4% and 46.9% respectively). The reduction in ratio of male and female fetuses was not statistically significant, without clear dose relation and was within historical control range (53.0% for 70 litters; min–max per litter, 14–79%) and considered to be non-adverse. The mean body weight of male fetuses in the 100 and 400 mg/kg bw/day dose groups was slightly decreased compared to the control vehicle group. This change (5.2% in both groups) was not statistically significant and considered non-adverse. The test item treatment did not affect the body weight of female fetuses and mean values for all fetuses.
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
The absolute and normalized anogenital distance in male and female fetuses was not
significantly changed in all dose groups.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The total affected fetuses were approximately the same among all groups.
In the test item treated groups, the following external malformations were revealed for single fetuses.
Umbilical hernia was observed in 3/231 (2 litters affected), 1/233. 0/243. 2/188 (2 litters affected) fetuses in the control, low, mid and high dose respectively. This observation has low incidence in the historical control population (2/878 fetuses, 2 litters affected) and was associated in the present study in most cases with generalized subcutaneous oedema (anasarca), which was observed in the approximately same incidence in all groups including control.
One fetus in the 100 mg/kg bw/day dose group had a malformed forepaw unilaterally. The absence of all carpals, metacarpals, and phalanges on this forelimb was shown. Although such a finding was not shown in the historical control population, this single not dose depended malformation was considered not test item related.
Cleft palate was found in a single fetus from the 25 mg/kg bw/day dose group. This fetus had generalized subcutaneous oedema, domed head, and umbilical hernia and was small in size. So, the cleft palate associated with complex findings in a single fetus in the low dose group was considered not test item related.

Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
The increase in the fetal incidence of delayed ossification, which was considered to be caused by the test item, was revealed for some skeletal structures starting from 25 mg/kg bw/day.
In the 25 mg/kg bw/day dose group, the increase in fetal incidence of unossified forelimb phalanges (70.1% versus 46.5%, p < 0.001), unossified 7th cervical vertebra (70.9% versus 57.9%, p < 0.05), incomplete ossification of caudal vertebrae (30.8% vesus 14.9%, p < 0.01) was observed.
In the 100 mg/kg bw/day dose group, the increase in fetal incidence of unossified forelimb phalanges (65.6% versus 46.5%, p < 0.001), increase in fetal and litter incidence of unossified 5th sternebra (13.9% versus 2.6%, p < 0.01, and 41.7% versus 13.0%, p < 0.05), in fetal incidence of unossified 6th sternebra (18.0% versus 8.8%, p < 0.05) were revealed.
In the 400 mg/kg bw/day group, incomplete ossification of interparietal skull bone was observed with fetal incidence of 7.5% (versus 1.8%, p < 0.05) and litter incidence of 30.4% (versus 8.7%). The incidence of unossified 6th sternebra was increased to 18.3% compared to 8.8% in the control group (p < 0.05). The increase in the incidence of unossified forepaw phalanges and delayed ossification in the caudal vertebrae was approximately the same as in low and medium dose groups. Moreover, in the high dose group, a statistically significant increase in the fetal incidence of an unossified first thoracic vertebra is recorded (7.5% versus 1.8%, p < 0.05).
All findings of delayed ossification are considered skeletal variations to indicate fetal immaturity and a transient stage in fetal development correlated to the slight decrease in body weight and are not thought to be adverse.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
No malformations of soft tissues were found in any observed fetuses.
In the 100 and 400 mg/kg bw/day dose group, a slight increase in the fetal incidence of dilatation of brain ventricles was observed (9.1% and 10.5% compared to 4.2% in the control group). This finding was dose-dependent and frequently found in small fetuses (8 of 11 fetuses with ventricles alteration at 100 mg/kg bw/day, and 5 of 10 fetuses at 400 mg/kg bw/day dose) and considered to be test item related. However, the effect was not statistically significant and is not thought to be adverse.
Other visceral findings were observed in single cases without dose-dependence or with approximately the same fetal and litter incidence among all groups, including control, and so they are considered not treatment-related.
Other effects:
not examined

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
Remarks on result:
other: Decreased number of fetuses per litter at 400 mg/kg bw/day which correlated to increased pre-implantation loss.

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
400 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
presumably yes

Any other information on results incl. tables

Table 1. SUMMARY BODY WEIGHT AND BODY WEIGHT GAIN DATA for pregnant females

GROUP:

Control

2 (25 mg/kg bw/day)

3 (100 mg/kg bw/day)

4 (400 mg/kg bw/day)

 

MEAN ± SD

MEAN ± SD

MEAN ± SD

MEAN ± SD

 

N=23

N=24

N=24

N=23

Gestation Day

Body weight, g

0

168 ± 15

167 ± 15

167 ± 15

168 ± 14

5

184 ± 16

184 ± 16

181 ± 16

186 ± 14

8

192 ± 16

192 ± 16

189 ± 17

193 ± 14

11

203 ± 15

203 ± 17

201 ± 16

204 ± 15

14

214 ± 17

214 ± 17

212 ± 16

214 ± 16

17

235 ± 18

231 ± 21

233 ± 18

226 ± 23

20

263 ± 20

258 ± 22

260 ± 21

251 ± 21

Period, gestation days

Body weight gain, %

5-8

4.4 ± 1.7

4.7 ± 2.4

4.6 ± 3.1

3.6 ± 1.8

5-11

10.8 ± 2.5

10.7 ± 2.6

11.0 ± 3.5

9.4 ± 2.5

5-14

16.8 ± 2.7

16.7 ± 2.8

17.5 ± 3.3

14.9 ± 2.8

5-17

28.0 ± 3.7

25.9 ± 6.7

28.8 ± 4.2

21.5 ± 9.4 (a)(c)(d)

5-20

43.4 ± 5.8

40.8 ± 8.1

43.7 ± 6.7

35.1 ± 8.3 (a)(b) (d)

(a) = Significantly different from 0 mg/kg bw/day  at 0.001 using repeated measures ANOVA Duncan test;

(b) = Significantly different from 25 mg/kg bw/day  at 0.001 using repeated measures ANOVA Duncan test;

(c) = Significantly different from 25 mg/kg bw/day at 0.01 using repeated measures ANOVA Duncan test;

(d) = Significantly different from 100 mg/kg bw/day  at 0.001 using repeated measures ANOVA Duncan test.

 

 

TABLE 2. SUMMARY DATA OF gravid Uterine weight and final maternal body weight

GROUP:

Control

2 (25 mg/kg bw/day)

3 (100 mg/kg bw/day)

4 (400 mg/kg bw/day)

 

MEAN ± SD

MEAN ± SD

MEAN ± SD

MEAN ± SD

 

N=23

N=24

N=24

N=23

Body weight at necropsy on gestation day 20, g

263 ± 20

258 ± 22

259 ± 21

251 ± 20

Gravid uterine weight, g

54.6 ± 12.3

51.9 ± 12.8

53.3 ± 8.0

45.1 ± 18.5 (a)

Final body weight without uterus, g

208 ± 19

206 ± 19

205 ± 18

205 ± 25

(a) = Significantly different from Control  at 0.05 using ANOVA Dunnett test.

TABLE 3. SUMMARY DATA OF corpora lutea number and pre-implantation loss

 

Historical Control

Control

25 mg/kg bw/day

100 mg/kg bw/day

400 mg/kg bw/day

 

(N=70)

(N=23)

(N=24)

(N=24)

(N=23)

Number of corpora lutea

 

 

 

 

 

Total, N

1102

280

290

293

276

MEAN ± SD

15.7 ± 3.0

12.2 ± 1.6

12.1 ± 1.4

12.2 ± 1.8

12.0 ± 2.2

Number of implantation sites

 

 

 

 

 

Total, N

918

246

247

255

202

MEAN ± SD

13.1 ± 2.2

10.7 ± 1.9

10.3 ± 2.3

10.6 ± 1.2

8.8 ± 3.2 (b)

Pre-implantation loss

 

 

 

 

 

Total, N

184

34

43

38

74 (a)

% of corpora lutea

16.7 %

12.1 %

14.8 %

13.0 %

26.8 % (a)

MEAN ± SD, N

2.6 ± 2.6

1.5 ± 1.7

1.8 ± 2.2

1.6 ± 1.7

3.2 ± 3.4 (b)

(a) Difference from control group 0 mg/kg bw/day with p < 0.01, Yates corrected Chi-square test

(b) Difference from control group 0 mg/kg bw/day with p < 0.05, ANOVA Dunnett test.

TABLE 4. SUMMARY DATA OF uterine content

   

Historical Control

Control

25 mg/kg bw/day

100 mg/kg bw/day

400 mg/kg bw/day

Number of treated females with confirmed mating

N

 

24

24

25

25

Number of pregnant females

N pregnant

70

23

24

24

23
   

 

        

Implantation sites

N

918

246

247

255

202

N per animal

MEAN ± SD

13.1 ± 2.2

10.7 ± 1.9

10.3 ± 2.3

10.6 ± 1.2

8.8 ± 3.2 (a)
   

 

  

 

 

 

Resorptions - Early

N

32

13

11

10

14

% of implantation sites

  

3.5

5.3

4.5

3.9

6.9

N per animal

MEAN ± SD

0.5 ± 0.7

0.6 ± 0.7

0.5 ± 0.5

0.4 ± 0.8

0.6 ± 0.7
   

 

  

 

 

 

Resorptions - Late

N

8

2

2

2

0

% of implantation sites

  

0.9

0.8

0.8

0.8

0.0

N per animal

MEAN ± SD

0.1 ± 0.4

0.1 ± 0.1

0.1 ± 0.3

0.1 ± 0.3

0.0 ± 0.0
   

 

  

 

 

 

Post-implantation Loss

Total

40

15

14

12

14

% of implantation sites

  

4.4

6.1

5.7

4.7

6.9

N per animal

MEAN ± SD

0.6 ± 1.0

0.7 ± 0.6

0.6 ± 0.7

0.5 ± 0.8

0.6 ± 0.7

Number of females with post-implantation loss

N / N pregnant

28 / 70

13 / 23

12 / 24

8 / 24

11 / 23

 

 

 

 

 

 

 

Total Fetuses

N

878

231

233

243

188

Alive

%

100.0

100.0

100.0

100.0

100.0

Dead

%

0.0

0.0

0.0

0.0

0.0

No. per animal

MEAN ± SD

12.5 ± 2.3

10.0 ± 1.9

9.7 ± 2.4

10.1 ± 1.7

8.2 ± 3.3 (a)

(a) Difference from control group 0 mg/kg bw/day with p < 0.05, ANOVA Dunnett test.

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:
Under the conditions of the present study the test substance did not induce any treatment-related biologically relevant malformations in the developing unborn organism in the presence of maternal toxicity. Therefore, the test substance does not meet the criteria for classification for prenatal developmental toxicity according to Regulation (EC) No 1272/2008. The available data is thus conclusive but not sufficient for classification.
Executive summary:

A prenatal developmental toxicity study was conducted to evaluate the potential effects of the test item, 2,4,6-trimethylphenol, on pregnancy and on embryo-fetal development in rats following daily oral (gavage) administration at doses 25, 100 and 400 mg/kg body weight (bw)/day from implantation to the day prior to scheduled caesarean section (Day 5 to Day 19 post-mating inclusive) (Khokhlova, 2022). The study was carried out in accordance to the OECD Guideline 414: Prenatal Developmental Toxicity Study (2018) in GLP conditions. The test item, 2,4,6-trimethylphenol, in the vehicle (corn oil), was administered by gavage once daily to Sprague-Dawley (SD) female rats from Day 5 to Day 19 post-mating (inclusive). Three animal groups were received the test item at dose levels of 25, 100 or 400 mg/kg bw/day. A concurrent vehicle control group has received the vehicle (corn oil) on a comparable regimen and in the same volume of 5 mL/kg bw. Each group consisted of 24 - 25 females with confirmed mating. All females were observed twice daily for mortality and morbidity and signs of toxicity following dose administration. Body weights and food consumption were recorded at three-day intervals. On post-mating Day 20, the dams were sacrificed and subjected to a macroscopic examination and enumeration of corpora lutea. The selected organs were weighed, and the weight of the thyroid gland, histopathological assessment of the thyroid gland, and assay of serum concentration of thyroxin (T4), triiodothyronine (T3), and thyroid-stimulating hormone (TSH) were conducted in dams to observe pathological changes in thyroid function. Gravid uteri including cervix were weighed, and uteri content was examined to record implantation sites, early and late resorptions, dead and live fetuses. The fetuses were weighed, sexed with measurement of anogenital distance (AGD), and submitted to external examination. Approximately half of the fetuses from each litter were subjected to a detailed examination of soft tissue by serial sectioning after fixation in Bouin’s solution, while the other half underwent detailed skeletal examination following staining of bone with alizarin red and cartilage with alcian blue. The fetal findings were classified as malformations or variations. Fetal incidence, litter incidence, and affected fetuses per litter were calculated for external, visceral, and skeletal alterations. The reproductive tract was examined with particular attention, and external sex was compared with internal (gonadal) sex in all fetuses; any incomplete testicular descent was noted in male fetuses. No mortality was observed in treated or control dams. Temporary prone position was recorded for one female after each 400 mg/kg bw/day dose administration starting from Day 13; hypersalivation was recorded in this female on Day 13. These effects were considered to be treatment related and adverse. There were no treatment related effects on bodyweight or food consumption. There were no treatment related effects observed following gross necropsy or following histopathology of the thyroid glands. There were no significant changes in the T3, T4, and TSH hormones level in test-treated females compared to the control vehicle group. The mean number of implantation sites was decreased in the 400 mg/kg bw/day treated females (by 17.8% compared to control group, p < 0.05), and the pre-implantation loss (%) was statistically higher when compared to the control group (26.8% versus 12.1%, p < 0.01) and higher than the historical control value of 16.7%. The mean number of fetuses per uterus was reduced in the 400 mg/kg bw/day dose group (18%, p < 0.05, compared to control group) which correlated with the decrease in the number of implantation sites at this dose. This finding was considered to be adverse. The percentage and the mean number of early and late resorptions and total postimplantation loss were not statistically changed compared to the control vehicle group. The number of females with post-implantation loss was approximately the same in all groups. There were no post-implantation embryo/fetal loss. There were no fetuses with test item related external, visceral or skeletal malformations. The body weight of male fetuses were non-significantly decreased and were associated with the delayed ossification in interparietal skull bone, forelimb phalanges, 6th sternebrae, 1st thoracic and caudal vertebrae. Incidences of delayed ossification in these bones were increased starting from the 25 mg/kg bw/day dose correlating to the slight decrease in fetal body weight and with values that were within the historical control data range or slightly exceeding it. Effects indicated fetal immaturity and considered to be variations and therefore not adverse.
It was concluded from this study that the dosage of 100 mg/kg bw/day was the maternal no observed adverse effect level (NOAEL). Due to the reduced number of fetuses per uterus correlated to the increased pre-implantation loss on 400 mg/kg bw/day dose, the NOAEL for embryo-fetal survival and development is 100 mg/kg bw/day.