Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Screening for reproductive/developmental toxicity: Data waiving (study scientifically not necessary): According to REACH Annex VIII, column 2, the study does not need to be conducted because a pre-natal developmental toxicity study is available.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because a pre-natal developmental toxicity study is available
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
According to REACH Annex VIII, column 2, the study does not need to be conducted because a pre-natal developmental toxicity study is available.
Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

Pre-natal developmental toxicity: Key study. Test method OECD 414, GLP study. Methyleugenol did not affect prenatal viability or the incidence of fetal malformations (external, visceral or skeletal) at any dose. The maternal toxicity LOAEL was 80 mg/kg/day based on increased liver weight. The developmental toxicity LOAEL was 500 mg/kg/day based on reduced fetal body weight and mildly delayed skeletal ossification (skeletal variation). Thus, the developmental toxicity NOAEL was 200 mg/kg/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 April 1998 – 10 June 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Remarks:
(Food and Drug Administration (FDA) Good Laboratory Practice)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
(Crl:CDR(SD)BR VAF/PlusR)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Inc., Raleigh, NC)
- Weight at study initiation: 225 to 275 g on gd 0
- Housing: Confirmed-mated females and ten additional sentinel females were individually housed in solid-bottom polycarbonate cages with stainless steel wire lids (Laboratory Products, Rochelle Park, NJ) and certified Sani-Chip® hardwood cage litter (P.J. Murphy, Montville, NJ).
- Diet (e.g. ad libitum): Purina Certified Rodent Chow® (#5002; PMI, St.Louis, MO), ad libitum.
- Water (e.g. ad libitum): tap water, ad libitum.
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 70.5-73.7ºF. 100% of the hourly measurements were within the target range (i.e., 69-75ºF).
- Humidity (%): 46.7-63.8%. 100% of the hourly measurements were within the target range (i.e., 35-65% RH).
- Photoperiod (hrs dark / hrs light): 12 light / 12 hours dark
Route of administration:
oral: gavage
Vehicle:
other: 0.5% aqueous methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: During the study, two sets of dose formulations were prepared. Dose formulations were stored in sealed, amberglass bottles at room temperature, and mixed well prior to dosing. Formulations were administered within the period of proven stability (21 days) and were based on a weight taken just before daily dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not reported.
- Concentration in vehicle: 0, 16, 40, 100 mg/mL, giving dose levels of 0, 80, 200, 500 mg/kg/day.
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Aliquots of the two sets of dose formulations were submitted for verification of concentration before each period of use. Measured concentrations were within 100.8-102.3% of the theoretical concentration.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: female rats were housed overnight with adult males from the same strain and supplier.
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Further matings after two unsuccessful attempts yes (three consecutive breeding dates)
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: The morning on which sperm were found in the vaginal lavage (Hafez, 1970) was designated as gd 0.
Duration of treatment / exposure:
Days 6 – 19 of gestation
Frequency of treatment:
Daily
Duration of test:
From day 0 to day 20 of gestation, when dams were euthanized.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
(vehicle control group)
Dose / conc.:
80 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 timed-mated females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on a screening study in which CD® rats were treated by gavage with 0, 31.25, 62.5, 125, 250 or 500 mg /kg body weight/day on gestational days (gd) 6 through 19. Evidence of developmental toxicity was limited to a decreasing dose-response trend for average fetal body weight per litter. Maternal toxicity was noted at >125 mg/kg/day (NTP, 1998).
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily. Females were observed for clinical condition at least once/day on gd 0 to 5. From gd 6 through 19, females were observed for clinical condition and signs of toxicity at dosing. In addition, postdosing observations were usually made between 1-2.5 hours after administration of the daily dose. On gd 20, females were observed for clinical condition at weighing and at scheduled termination.

BODY WEIGHT: Yes
- Time schedule for examinations: Maternal body weights were recorded on the mornings of gd 0, 6-20, and immediately following sacrifice on gd 20.

FOOD CONSUMPTION: Yes, food consumption was monitored during the study, with measurements on the mornings of gd 0, 6, 9, 12, 15, 18, 19 and 20.

WATER CONSUMPTION: Yes, water consumption was monitored during the study, with measurements on the mornings of gd 0, 6, 9, 12, 15, 18, 19 and 20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: the body, liver, and gravid uterus of each timed-mated female were weighed. Thoracic and abdominal cavities were examined. Maternal livers were saved in 10% neutral buffered formalin. No maternal gross pathology was observed at necropsy, and therefore no additional maternal tissues were saved.

-OTHER: Formalin-fixed livers from 10 randomly selected dams per group were trimmed, sectioned and stained with hematoxylin and eosin (H&E). Sections were mounted on slides and submitted to for light microscopic examination.
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 and late resorptions: Yes
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 (as part of soft tissue examination)


Statistics:
Quantitative continuous data were compared among treatment groups by parametric statistical tests whenever Bartlett’s test for homogeneity of variance was not significant. When Bartlett’s test indicated a lack of homogeneity (p<0.001), nonparametric statistical tests were applied.
General Linear Models (GLM) procedures were applied to the Analyses of Variance (ANOVA) and the Tests for Linear Trend. Prior to GLM analysis, an arcsine-square root transformation was performed on all litter-derived percentage data. ANOVA was weighted according to litter size. When a significant (p<0.05) main effect for dose occurred, Dunnett’s Multiple Comparison Test was used. A one-tailed test (i.e., Dunnett’s Test) was used for all pairwise comparisons to the vehicle control group, except that a two-tailed test was used for maternal body and organ weight, maternal feed and water consumption, fetal bw, and % males per litter. Nonparametric tests applied to continuous variables included the Kruskal-Wallis one-way ANOVA and, if significant (p<0.05), the Mann-Whitney U test. A one-tailed Mann-Whitney U test was used for all parameters, except that maternal and fetal bw, as well as maternal feed and water consumption were examined in a two-tailed test. Jonckheere's test for k independent samples was used to identify significant dose-response trends.
Nominal scale measures were analyzed by Chi-Square Test for Independence for differences among treatment groups and by the Cochran-Armitage Test for Linear Trend on Proportions. When Chi-Square revealed significant (p<0.05) differences among groups, then a one-tailed Fisher's Exact Probability Test was used.
The alpha level for each statistical comparison was 0.05, and the significance levels for trend tests and pairwise comparisons (one- or two-tailed) were reported as p<0.05 or p<0.01.
Historical control data:
Historical control data from the laboratory were used for comparisons and appropiate summaries were attached to the the study report.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Sedation was noted in the high dose group with 3/22 females lethargic and 8/22 prone after dosing on gd 6.
Rooting in the cage bedding after dosing indicated an aversion to the taste, odor, or other sensory properties of the dose formulation. Rooting was observed in 0, 10, 14, and 19 females from the control through high-dose groups, respectively, on gd 11.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No treatment-related maternal deaths occurred in this study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Maternal body weight exhibited significant dose-response trends on gd 9, 12, 15, 18, 19 and 20. Reductions in body weight were statistically significant at the high dose on these days. Maternal weight gain was affected primarily during the early (gd 6-9) and late (gd 15-18) portions of the treatment period, with significant reductions at the highest dose.
Maternal weight gain across the entire treatment period (gd 6 to 20) also showed a significant reduction at the high dose, while weight gain during gestation and corrected weight gain were reduced in both the mid- and high-dose groups.
Gravid uterine weight was equivalent among groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Maternal relative feed consumption (g/kg/day) was occasionally elevated (104-107% of control intake) during the treatment period. Specifically, feed consumption was elevated from gd 12 to 15 for the low-dose group, and from gd 12 to 15 and 15 to 18 for the mid-dose group. More importantly, maternal relative feed consumption was significantly decreased at the high dose: 61% of control consumption during early treatment (gd 6-9), 83% during late treatment (gd 18-19), and 91% for the treatment period as a whole (gd 6-20).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
During early treatment (gd 6-9), maternal water consumption was reduced at the high dose, but was not affected thereafter.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry 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, treatment-related
Description (incidence and severity):
Maternal liver weight (absolute and relative to body weight) showed an increasing dose-related trend on gd 20, and these weights were significantly elevated above controls in all treatment groups.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Hepatocellular necrosis occurred in two females (1/10 controls and 1/10 high dose dams) but in the absence of a dose-related pattern.
Treatment-related lesions included (a) hepatocellular cytoplasmic vacuolization noted in 6/10 and 10/10 dams in the mid- and high-dose groups, respectively, and (b) hepatocellular degeneration noted in 2/10 high-dose dams.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Description (incidence and severity):
No abortions were registered either in treated groups or in the control group.
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
The percent resorptions per litter and the percent litters with one or more resorptions did not differ among groups.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no reported late fetal deaths in this study.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
At termination, the pregnancy rates ranged from 72-92% per group. Lower than expected pregnancy rates were probably due to the use of novice male breeders for this study, and the lowest pregnancy rate occurred in the control group.
Other effects:
no effects observed
Key result
Dose descriptor:
LOAEL
Effect level:
80 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
organ weights and organ / body weight ratios
Remarks on result:
other: Effects observed at the lowest dose tested.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Average fetal body weight per litter in treated groups was decreased in a dose-related manner (95, 96 and 86% of the average control weight). This reduction was statistically significant at the high dose, and the dose response was similar for males, females or both sexes combined.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no effects on live fetuses.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no effects on fetal sex ratio (percent male fetuses per litter).
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Average live litter size in treated groups was between 104-109% of the control mean, with no statistically significant difference among groups.
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
There were no external malformations or variations observed in this study.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
There were no skeletal malformations observed in this study.
The skeletal variations identified in this study (bipartite ossification centers with normal or dumbbell-shaped cartilage) occurred in all treatment groups, and the incidence showed no apparent relation to dose. Variations of the thoracic centra also occur commonly in the CD® rat (RTI unpublished, 1989-1997).
Lumbar I rib is also a common finding in this species/strain (RTI unpublished, 1989-1997). In this study, the incidence of rudimentary Lumbar I rib(s) appeared to be dose related, i.e., 0.8% (1/126), 1.5% (2/134), 1.8% (3/166), and 5.0% (8/160) for the control through high-dose groups, respectively. It is worth noting that the observed incidence for each group was well within the previously observed range for individual study control groups (0-12.7%). Thus, the incidence of rudimentary Lumbar I rib appears to be equivocally related to test item exposure, even at the high dose in this study.
Unossified sternebra(e) occurred in 2.4% (3/126), 3.7% (5/134), 2.4% (4/166), and 11.9% (19/160) of the fetal skeletons examined. The increased incidence at 500 mg/kg/day reflects a mild developmental delay, consistent with the observed reduction in average fetal body weight at the same dose.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The incidence of visceral malformations did not differ among treatment groups. Except for one case of mild hydrocephaly in the mid-dose group, the only reported visceral malformations were hydronephrosis and hydroureter.
Historical control data from laboratory, as well as reports from other laboratories (Van Winkle et al., 1988; Chandra and Frith, 1993) clearly indicate that these renal anomalies are commonly found in Sprague-Dawley-derived rats, and that their occurrence may have a hereditary basis (Hume et al., 1980).
The most common visceral variation (i.e., distended ureter), was noted in 2.1-9.4% of foetuses examined/group, but the incidence was not dose related.
Other visceral variations included slightly enlarged lateral ventricles (four fetuses in the low dose group), enlarged nasal sinuses (one fetus each in the control through mid-dose groups), and extra liver tissue (one control fetus). Based on the absence of a dose-response relationship, these findings did not appear to be related to test item exposure.
Another visceral variation (i.e., agenesis of the innominate artery) occurred in 0.8% (1/125), 0.7% (1/138), 0.6% (1/171) and 2.5% (4/159) of fetuses in the control through high-dose groups, respectively. By comparison, the historical control incidence was 0.05% (2/4016 fetuses), and the highest incidence for an individual study control group was 0.6% (1/159) (RTI unpublished, 1989-1997). Other sources reported a comparable average control group incidence (i.e., 0.5% per study) and a wider range for individual study controls (0-3.4%) based on 154 studies and 22,892 fetuses (Charles River, 1993). Thus, individual groups in this study fell within the previously observed control group incidence for agenesis of the innominate artery in the CD® rat. Furthermore, post hoc statistical analyses did not find any significant effects of the test item on the incidence of this variation.
Other effects:
no effects observed
Key result
Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
Remarks on result:
other: Effects observed at the highest dose tested.
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
Remarks on result:
other: Effects observed at the highest dose tested.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table 1. Maternal Toxicity in CD Rats Exposed to Methyleugenol on Gestational Days 6 Through 19.

 

Methyleugenol (mg/kg/day)

 

0

80

200

500

Maternal Pregnancy Status

 

 

 

 

No. treated

25

25

25

25

No. removed

0

1

0

1

No. dead or euthanized

0

0

0

0

No. (%) pregnant at sacrifice

18(72)

18(75)

23(92)

22(92)

Maternal Body Weight Changes (g)a,b

 

 

 

 

Gestation wt. gain (gd 0 to 20)

129.9±3.8§§

127.8±2.9

114.6±4.3*

96.9±5.5**

Treatment wt. gain (gd 6 to 20)

107.1±3.9§§

109.4±2.8

95.1±3.7

73.4±5.8**

Corrected wt. Gainc

50.2±2.9§§

43.5±2.5

32.2±2.4**

23.8±3.3**

Gravid uterine wt.

79.7±3.0

84.4±2.7

82.5±3.7

73.0±4.1

Maternal Organ Weightsa

 

 

 

 

Liver: Absolute (g)

15.95±0.28§§

16.97±0.26*

17.50±0.27**

17.69±0.56*

       Relative (% body wt.)d

4.18±0.06§§

4.53±0.05**

4.81±0.05**

5.12±0.13**

Maternal Histopathology

 

 

 

 

Hepatocellular Lesionsf

 

 

 

 

Necrosis, minimal

1

0

0

1

Cytopasmic vacuolization,

 

 

 

 

            (periportal), minimal

0

0

6

7

             mild

0

0

0

3

Degeneration, minimal

0

0

0

2

Maternal Feed Consumptiona,e

 

 

 

 

Pretreatment period (gd 0 to 6)

 

 

 

 

      Absolute (g/day)

22.2±0.7

22.2±0.6

22.8±0.5

22.3±0.4

      Relative (g/kg/day)

83.3±2.5

85.1±1.5

86.4±1.3

85.1±1.1

Treatment period (gd 6 to 20)

 

 

 

 

      Absolute (g/day)

23.9±0.6§§

24.5±0.4

24.0±0.5

19.8±0.6**

      Relative (g/kg/day)

71.7±1.1§§

75.2±0.9

74.9±1.0

65.2±1.7**

Maternal Water Consumptiona,e

 

 

 

 

Pretreatment period (gd 0 to 6)

 

 

 

 

      Absolute (g/day)

34.2±1.9

33.3±1.2

35.5±1.8

33.3±1.2

      Relative (g/kg/day)

128.2±6.8

127.3±3.7

134.4±6.3

127.3±4.6

Treatment period (gd 6 to 20)

 

 

 

 

      Absolute (g/day)

43.1±2.1§

41.1±1.0

42.7±2.3

36.8±1.8

      Relative (g/kg/day)

129.6±5.8

126.2±2.5

133.3±6.5

120.2±5.3

a Includes all dams pregnant at sacrifice; mean ± SEM; gd = gestational day.

b Body weights were recorded in the morning of each designated gestational day.

c Weight change during gestation minus gravid uterine weight.

d Calculated using body weight at the time of sacrifice on gd 20.

e Body weights were recorded in the morning of each designated gestational day. Relative food and water consumption (g/kg/day) were calculated using these weights.

f Number of livers affected; ten livers were examined per group.

§ p<0.01; Test for Linear Trend

* p<0.05; Dunnett’s test

** p<0.01; Dunnett’s test.

Table 2. Developmental Toxicity in CD Rat Fetuses Following Maternal Exposure to Methyleugenol on Gestational Days 6 Through 19.

 

Methyleugenol (mg/kg/day)

 

0

80

200

500

All Littersa,b

18

18

23

22

No. corpora lutea/dam

14.78±0.44

16.00±0.35

16.05±0.74

15.50±0.57

No. implantation sites/litter

14.44±0.55

15.56±0.42

15.22±0.74

14.95±0.79

% preimplantation loss/litter

4.47±1.85

3.26±1.78

6.78±2.45

6.46±3.00

% resorptions/litter

4.15±1.57

2.53±1.17

2.34±0.89

3.44±1.55

% litters with resorptions

39

28

26

36

% late deaths/litterc

0

0

0

0

% litters with late deathsc

0

0

0

0

 

 

 

 

 

Live Littersb,d

18

18

23

22

No. live fetuses/litter

13.94±0.65

15.17±0.46

14.83±0.71

14.55±0.82

Avg. male fetal body wt./litter (g)

3.73±0.12‡

3.58±0.05

3.60±0.04

3.22±0.11*

Avg. female fetal body wt./litter (g)

3.60±0.12‡

3.38±0.05

3.42±0.04

3.09±0.10*

% male fetuses/litter

46.73±2.38

47.33±3.03

49.20±2.64

47.27±2.57

% externally malformed

fetuses/litterc

0

0

0

0

% viscerally malformed

fetuses/litter

3.67±2.54

4.13±2.41

4.32±1.71

5.38±2.12

% skeletally malformed

fetuses/litterc

0

0

0

0

% malformed fetuses/litter

1.78±1.24

2.09±1.21

2.25±0.89

2.67±1.05

% fetuses with unossified

sternebra/littere

4.50±3.74§

4.10±2.57

2.66±2.21

11.69±4.04*

a Includes all dams pregnant at sacrifice; litter size = no. implantation sites per dam.

b Reported as the mean ± SEM.

c For parameters with a zero incidence in every group, statistical analyses were not performed.

d Includes only dams with live fetuses; litter size = no. live fetuses per dam.

e Unossified sternebra (I, II, III and/or IV only) is classified as a variation.

‡ p < 0.01 Jonckheere’s Test

* p < 0.01 Mann-Whitney U Test or Dunnett’s Test

§ p < 0.05 Test for Linear Trend

Table 3. Morphological Abnormalities in CD Rat Fetuses: Listing by Defect Type (a)

 

Methyleugenol (mg/kg/day)

 

0

80

200

500

ANY MALFORMATIONS

 

 

 

 

Total No. of Fetuses Examined for Any Malformationsb

251

273

341

320

No. of Fetuses with Any Malformationsc

5

5

8

9

% Fetuses with Any Malformations

2.0%

1.8%

2.3%

2.8%

Total No. of Litters Examined for Any Malformationsd

18

18

23

22

No. of Litters with Any Malformationse

2

3

6

6

% Litters with Any Malformations

11.1%

16.7%

26.1%

27.3%

EXTERNAL MALFORMATIONS

 

 

 

 

Total No. of Fetuses Examined for External Malformationsb

251

273

341

320

No. of Fetuses with External Malformationsc

0

0

0

0

% Fetuses with External Malformations

0.0%

0.0%

0.0%

0.0%

Total No. of Litters Examined for External Malformationsd

18

18

23

22

No. of Litters with External Malformationse

0

0

0

0

% Litters with External Malformations

0.0%

0.0%

0.0%

0.0%

VISCERAL MALFORMATIONS

 

 

 

 

Total No. of Fetuses Examined for Visceral Malformationsb

125

138

171

159

No. of Fetuses with Visceral Malformationsc

5

5

8

9

% Fetuses with Visceral Malformations

4.0%

3.6%

4.7%

5.7%

Total No. of Litters Examined for Visceral Malformationsd

18

18

23

22

No. of Litters with Visceral Malformationse

2

3

6

6

% Litters with Visceral Malformations

11.1%

16.7%

26.1%

27.3%

Hydrocephaly: Mild

 

 

1(1)

 

Hydronephrosis: Bilateral

4(2)

 

3(2)

5(4)

Left

 

2(2)

1(1)

2(2)

Right

 

2(2)

2(2)

2(2)

Hydroureter: Bilateral

2(1)

 

2(1)

1(1)

Left

 

3(2)

1(1)

2(1)

Right

1(1)

2(2)

2(2)

1(1)

SKELETAL MALFORMATIONS

 

 

 

 

Total No. of Fetuses Examined for Skeletal Malformationsb

126

134f

166g

160f

No. of Fetuses with Skeletal Malformationsc

0

0

0

0

% Fetuses with Skeletal Malformations

0.0%

0.0%

0.0%

0.0%

Total No. of Litters Examined for Skeletal Malformationsd

18

18

23

22

No. of Litters with Skeletal Malformationse

0

0

0

0

% Litters with Skeletal Malformations

0.0%

0.0%

0.0%

0.0%

ANY VARIATIONS

 

 

 

 

Total No. of Fetuses Examined for Any Variationsb

251

273

341

320

No. of Fetuses with Any Variationsc

39

39

42

53

% Fetuses with Any Variations

15.5%

14.3%

12.3%

16.6%

Total No. of Litters Examined for Any Variationsd

18

18

23

22

No. of Litters with Any Variationse

15

17

20

19

% Litters with Any Variations

83.3%

94.4%

87.0%

86.4%

EXTERNAL VARIATIONS

 

 

 

 

Total No. of Fetuses Examined for External Variationsb

251

273

341

320

No. of Fetuses with External Variationsc

0

0

0

0

% Fetuses with External Variations

0.0%

0.0%

0.0%

0.0%

Total No. of Litters Examined for External Variationsd

18

18

23

22

No. of Litters with External Variationse

0

0

0

0

% Litters with External Variations

0.0%

0.0%

0.0%

0.0%

VISCERAL VARIATIONS

 

 

 

 

Total No. of Fetuses Examined for Visceral Variationsb

125

138

171

159

No. of Fetuses with Visceral Variationsc

14

9

18

13

% Fetuses with Visceral Variations

11.2%

6.5%

10.5%

8.2%

Total No. of Litters Examined for Visceral Variationsd

18

18

23

22

No. of Litters with Visceral Variationse

8

6

12

11

% Litters with Visceral Variations

44.4%

33.3%

52.2%

50.0%

Enlarged Lateral Ventricle (Full): Bilateral

 

1(1)

 

 

Enlarged Lateral Ventricle (Half): Bilateral

 

2(2)

 

 

Left

 

1(1)

 

 

Enlarged Nasal Sinus

1(1)

1(1)

1(1)

 

Agenesis of the Innominate Artery

1(1)

1(1)

1(1)

4(3)

Extra Piece of Liver Tissue on Median Liver Lobe

1(1)

 

 

 

Distended Ureter: Bilateral

6(4)

1(1)

7(6)

4(4)

Left

3(3)

1(1)

8(8)

5(5)

Right

2(2)

1(1)

1(1)

1(1)

SKELETAL VARIATIONS

 

 

 

 

Total No. of Fetuses Examined for Skeletal Variationsb

126

134f

166g

160f

No. of Fetuses with Skeletal Variationsc

25

30

24

40

% Fetuses with Skeletal Variations

19.8%

22.4%

14.5%

25.0%

Total No. of Litters Examined for Skeletal Variationsd

18

18

23

22

No. of Litters with Skeletal Variationse

11

13

14

16

% Litters with Skeletal Variations

61.1%

72.2%

60.9%

72.7%

Unossified Sternebra (I, II, III and/or IV only)

3(2)

5(3)

4(2)

19(9)

Rib on Lumbar I: Bilateral Rudimentaryh

1(1)

1(1)

1(1)

6(2)

                   Left Rudimentaryh

 

 

2(2)

2(2)

                   Right Rudimentaryh

 

1(1)

 

 

Normal Cartilage, Bipartite Ossification Center: Thoracic Centrum

16(7)

19(7)

15(7)

10(7)

Dumbbell Cartilage, Bipartite Ossification Center: Thoracic Centrum

5(3)

5(5)

2(2)

4(1)

a A single fetus may be represented more than once in listing individual defects. Data are presented as the number of fetuses (number of litters).

b Only live fetuses were examined.

c Fetuses with one or more malformations/variations.

d Includes only litters with live fetuses.

e Litters with one or more malformed/variant fetuses.

f One fetus did not have a skeletal exam due to a problem in the staining process which made it unreadable.

g Four fetuses did not have skeletal exams due to a problem in the staining process which made them unreadable.

h Rib on Lumbar I is classified as “rudimentary” when the ossified portion is less than one-half the length of Rib XIII.

Conclusions:
Under the present test conditions, methyleugenol did not affect prenatal viability or the incidence of fetal malformations (external, visceral or skeletal) at any dose. The maternal toxicity LOAEL was 80 mg/kg/day based on increased liver weight. The developmental toxicity LOAEL was 500 mg/kg/day based on reduced fetal body weight and mildly delayed skeletal ossification (skeletal variation). Thus, the developmental toxicity NOAEL was 200 mg/kg/day.

Executive summary:

A pre-natal developmental toxicity test was performed with methyleugenol (MEUG) following a method similar to OECD Guideline 414 and in compliance with GLP. Groups of 25 timed-mated Sprague-Dawley rats were administered 0, 80, 200 or 500 mg/kg/day of MEUG diluted in vehicle (0.5% aqueous methylcellulose) by gavage on gestation days 6–19. On Day 20 of gestation, the animals were killed and both dams and fetus were examined.Confirmed pregnancy rates were 72-92% per group. No treatment-related maternal deaths occurred in this study. Clinical signs included evidence of maternal sedation at 500 mg/kg/day, and aversion to dosing at all doses. Maternal body weight gain was reduced at 200 and 500 mg/kg/day. Gravid uterine weight was not affected. Increased maternal liver weight (absolute and relative) was treatment related and significant at all doses. Ten randomly selected maternal livers per group were submitted for histopathology. Hepatocellular cytoplasmic vacuolization was noted at≥200 mg/kg/day and hepatocellular degeneration at 500 mg/kg/day. Average fetal body weight per litter was significantly reduced at the high dose (86% of the average control weight). MEUG did not affect any indices of prenatal viability. Live litter size in MEUG-treated groups was 104-109% of the control mean. Incidences of fetal morphological anomalies were statistically equivalent among groups, except for an increased incidence of unossified sternebrae, a skeletal variation, at the high dose. According to these results, the maternal toxicity LOAEL was 80 mg/kg/day based on increased liver weight, and thus the maternal toxicity NOAEL was not determined. Also, the developmental toxicity LOAEL was 500 mg/kg/day based on intrauterine growth retardation and mildly delayed skeletal ossification, and the developmental toxicity NOAEL was 200 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study has a Klimisch score = 2
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Pre-natal developmental toxicity: Key study. A pre-natal developmental toxicity test was performed with methyleugenol (MEUG) following a method similar to OECD Guideline 414 and in compliance with GLP. Groups of 25 timed-mated Sprague-Dawley rats were administered 0, 80, 200 or 500 mg/kg/day of MEUG diluted in vehicle (0.5% aqueous methylcellulose) by gavage on gestation days 6–19. On Day 20 of gestation, the animals were killed and both dams and fetus were examined.Confirmed pregnancy rates were 72-92% per group. No treatment-related maternal deaths occurred in this study. Clinical signs included evidence of maternal sedation at 500 mg/kg/day, and aversion to dosing at all doses. Maternal body weight gain was reduced at 200 and 500 mg/kg/day. Gravid uterine weight was not affected. Increased maternal liver weight (absolute and relative) was treatment related and significant at all doses. Ten randomly selected maternal livers per group were submitted for histopathology. Hepatocellular cytoplasmic vacuolization was noted at≥200 mg/kg/day and hepatocellular degeneration at 500 mg/kg/day. Average fetal body weight per litter was significantly reduced at the high dose (86% of the average control weight). MEUG did not affect any indices of prenatal viability. Live litter size in MEUG-treated groups was 104-109% of the control mean. Incidences of fetal morphological anomalies were statistically equivalent among groups, except for an increased incidence of unossified sternebrae, a skeletal variation, at the high dose. According to these results, the maternal toxicity LOAEL was 80 mg/kg/day based on increased liver weight, and thus the maternal toxicity NOAEL was not determined. Also, the developmental toxicity LOAEL was 500 mg/kg/day based on intrauterine growth retardation and mildly delayed skeletal ossification, and the developmental toxicity NOAEL was 200 mg/kg/day.

Justification for classification or non-classification

Based on the available information, the substance is not classified for toxicity to reproduction in accordance with CLP Regulation (EC) no 1272/2008.

Additional information