2-(2-ethoxyethoxy)ethyl acetate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 23, 2020 to August 13, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2018-06-25

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-ethoxyethoxy)ethyl acetate

EC Number:

203-940-1

EC Name:

2-(2-ethoxyethoxy)ethyl acetate

Cas Number:

112-15-2

Molecular formula:

C8H16O4

IUPAC Name:

2-(2-ethoxyethoxy)ethyl acetate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Production lot
- Lot/batch number of test material: P230220120

- Expiration date of the lot/batch: 2023-04-29

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Cool, well ventilated place
- Stability under storage conditions: Stable
- Stability under test conditions: Stable

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Breeding Facility, Jai Research Foundation
- Age at study initiation: 12 - 14 weeks
- Weight at study initiation: 198.9 - 249.1 g
- Fasting period before study: No
- Housing: Mated females individually caged in solid floor polypropylene rodent cages with stainless steel top grill
- Diet: Teklad certified global 14% protein rodent diet, ad libitum
- Water: Water cleaned and filtered by reverse osmosis, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 deg C
- Humidity (%): 30 - 70%
- Air changes (per hr): > 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 2020-11-09 To: 2021-05-28

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Daily, prior to dosing, and utilised within 4 hours of peparation

VEHICLE
- Justification for use and choice of vehicle: Water was used as the vehicle as the test item was readily miscible in that medium
- Concentration in vehicle: 0, 10, 50 and 100 mg/mL
- Amount of vehicle: Dose volume of 10 mL/kg body weight administered

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Active ingredient content and homogeneity of formulations were analysed once before initiation of treatment and once during the treatment period. Three aliquots (from the upper, middle and lower layers) from each of the dose formulations and one aliquot from the vehicle control were sampled. Analysis was by GC/FID. On each analytical occasion, the mean concentration was determined and compared with the nominal value. The acceptance criteria were ± 10% deviation from the nominal value and %CV < 10.

Details on mating procedure:

- Impregnation procedure: Cohoused
- M/F ratio per cage: 1/1
- 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
- Any other deviations from standard protocol: No

Duration of treatment / exposure:

From Day 5 to Day 19 of gestation

Frequency of treatment:

Once daily

Duration of test:

20 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on outcome of a preliminary dose-ranging screen
- Rationale for animal assignment: Random

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for mortality/morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Gestation days 0, 3, 5, 8, 11, 14, 17 and 20

FOOD CONSUMPTION AND COMPOUND INTAK: Yes
- Time schedule for examinations: Gestation days 0, 3, 5, 8, 11, 14, 17 and 20
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes for periods - gestation days 0-3, 3-5, 5-8, 8-11, 11-14, 14-17 and 17-20

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Those with macroscopic abnormalities and the ovaries, uterus and cervix from all animals. Thyroid weight determined and the tissue preserved for possible histopathology.

OTHER:
At the time of terminal sacrifice on Day 20 of gestation, blood samples were taken from all surviving dams for thyroid hormone analysis. Serum thyroid hormones T3 and T4 were analysed using bioanalytical methods and TSH was analysed using an ELISA method.

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

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

Statistics:

Parametric data (body weight, body weight gain/change, food consumption, organ weight, and prenatal data etc.) was subjected to Shapiro-Wilk’s test for normality check wherever applicable, followed by Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. If the data did not meet the normality, data were transformed to check the normality again. If transformed data did not meet the normality check, the Kruskal-wallis/Mann Whitney test was performed to calculate significance. If the data did not meet the homogeneity of variance, statistical analysis was extended following the decision tree of Gad (Gad, S.C., 2007). Non-parametric data (mortality rate, pregnancy rate, foetal observations etc.) were analysed using Chi-square test.
Count data (foetal count, number of corpora lutea, number of implants, number of live foetuses, number of dead foetuses, number of pre-implantation loss, number of post-implantation loss, number of resorptions) were be subjected to non-parametric Kruskal-Wallis test.
AGD was normalised (the ratio of AGD to the cube root of body weight) and then subjected to statistical analysis.

Indices:

Number of male foetuses
Number of female foetuses
Body weight of male foetuses (g)
Body weight of female foetuses (g)
Foetus sex (based on ano-genital distance)
Ano-genital distance (mm)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical sign of toxicity was observed.

Mortality:

no mortality observed

Description (incidence):

No mortality and morbidity were observed.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weight and corrected body weight of the pregnant female rats were comparable between the control and treated groups up to a dose level of 500 mg/kg/day. At 1000 mg/kg/day, a statistically significant decrease in the mean body weight of the pregnant female rats was observed on gestation days 17 and 20. A decreased mean body weight for this group was also observed during gestation days 11 and 14 without statistical significance. A statistically significant decrease in day 20 corrected body weight was also noted for this group.

The mean body weight change of pregnant female rats was comparable between the control and test treated groups up to a dose level of 500 mg/kg/day. At 1000 mg/kg/day, a statistically significant decrease in mean body weight change was observed during gestation days 5-20.

Decreases in the mean body weight and body weight change are correlated with decreased mean food consumption and are considered as adverse effect of treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption of the pregnant female rats was comparable between the control and treated groups up to a dose level of 500 mg/kg/day. A statistically significant reduced mean food consumption was seen in 1000 mg/kg/day treated rats over gestation days 5-8, 11-14, and 5-20.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

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:

no effects observed

Description (incidence and severity):

Treatment did not lead to any alteration in absolute and relative weights of the thyroid gland.

Gross pathological findings:

no effects observed

Description (incidence and severity):

External and internal (gross) examination of terminally sacrificed female rats did not reveal any lesion of pathological significance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histological examination of the thyroid gland did not reveal any lesion in rats of the control as well as the high dose groups except ultimobranchial cyst/s present in a single female rat of the control group, which was considered as spontaneous or incidental in nature

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid Hormone Analysis
Serum TSH level was comparable with that of the control group. Statistically significant lower serum levels of T3 and T4 were observed in pregnant female rats belonging to 100 mg/kg/day dose group. Statistically significant lower serum level of T4 was also observed in pregnant female rats belonging to 1000 mg/kg/day dose group. These effects are considered as incidental and unrelated to the test item treatment due to lack of dose-dependency and absence of other supporting findings (Serum TSH level and other thyroid-related parameters such as thyroid weight and thyroid histopathology were comparable with that of the control group)

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

The mean absolute and relative uterine weight of the pregnant female rats were comparable between the control and the test item treated groups.
The mean numbers of corpora lutea, implantation sites, live foetuses, dead foetuses, resorptions (early, late, and total), pre-implantation loss, and post-implantation loss, the mean percent of live foetuses, dead foetuses, pre-implantation loss, post-implantation loss, and total resorptions were comparable between the control, and test item treated groups.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

2

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean body weight of male, female and total foetuses (male + female) was comparable between the control and treated groups

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Incidences of skeletal malformations were not observed in any of the dose groups.

Skeletal variations were noted as follows: A statistically significant decrease in the number of foetuses with 14th rib: extra ossification centre was observed in the 100 and 1000 mg/kg/day dose groups. This finding was a result of higher number of incidences in controls compared to treatment groups and has no toxicological relevance. A statistically significant increase in the number of foetuses with 2nd sternebrae: unossified was observed in the 500 mg/kg/day dose group. Due to the lack of dose dependency, this increase was considered as incidental and non-adverse. A statistically significant increase in the number of foetuses with xiphisternum: unossified was observed in the 500 mg/kg/day dose group. A statistically significant increase in the number of foetuses and litters with xiphisternum: unossified was observed in the 1000 mg/kg/day dose group. The increase in number of foetuses and litters with xiphisternum: unossified indicate a delayed ossification associated with marginal reduced foetal weights i.e., a delay in foetal development secondary to the maternal toxicity observed at this dose level rather than a direct effect on bone tissue. In addition, this increase in the number of foetuses and litters with Xiphisternum: Unossified was within the range of historical control data and therefore considered as a non-adverse effect of treatment.

Visceral malformations:

no effects observed

Description (incidence and severity):

No treatment-related visceral anomalies were observed in foetuses of the treatment groups up to the dose level of 1000 mg/kg/day. Some spontaneous findings such as situs inversus (one foetus from the 500 mg/kg/day dose group) and dilated ureter (two foetuses from the control and one foetus from the 500 mg/kg/day dose group) were observed.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Anogenital distance (AGD)
The mean anogenital distance of female foetuses was comparable between the control and treated groups.
A statistically significant increase in AGD of male foetuses was observed in the 1000 mg/kg bw/day dose group. However, due to absence of an observed effect in female foetuses and considering that the mean value of AGD of treated male foetuses is only 1.67% higher than that of controls, the finding was considered non-adverse (Robert, H. G., Jr. Joseph, F. H., Donald, G. S., John, F. K., Vincent, L. R., 1999: “Interpreting the Toxicologic Significance of Alterations in Anogenital Distance: Potential for Confounding Effects of Progeny Body Weights”, Reproductive Toxicology, 13: 383-390).

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Lack of effects

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Maternal Data

                                                                                                                     

Dose level	0 mg/kg bw/day		100 mg/kg bw/day		500 mg/kg bw/day		1000 mg/kg bw/day
Parameters	N	%	N	%	N	%	N	%
N° of estrus positive rats	25	100.00	25	100.00	25	100.00	25	100.00
N° of discarded after copulation	0	0.00	0	0.00	0	0.00	0	0.00
N° with premature delivery/abortion	0	0.00	0	0.00	0	0.00	0	0.00
Mortality	0	0.00	0	0.00	0	0.00	0	0.00
N° sacrificed under moribund condition	0	0.00	0	0.00	0	0.00	0	0.00
N° sacrificed at term (at 20^thday of Gestation)	25	100.00	25	100.00	25	100.00	25	100.00
N° non-pregnant females	1	4.00	0	0.00	4	16.00	0	0.00
N° pregnant females	24	96.00	25	100.00	21	84.00	25	100.00
N° with at least one viable foetus	24	96.00	25	100.00	21	84.00	25	100.00
N° with all fetuses viable	23	95.83	22	88.00	17	80.95	20	80.00
N° with at least one dead foetus	0	0.00	0	0.00	0	0.00	0	0.00
N° with all dead foetuses	0	0.00	0	0.00	0	0.00	0	0.00
N° with resorption	1	4.17	3	12.00	4	19.05	5	20.00
N° with complete resorption	0	0.00	0	0.00	0	0.00	0	0.00

Key: N = Number of observations

Maternal Body Weight (g)

 

Dose level	0 mg/kg bw/day			100 mg/kg bw/day			500 mg/kg bw/day			1000 mg/kg bw/day
Gestation Day	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
0	238.72	9.11	24	239.33	10.39	25	238.73	9.73	21	237.59	9.01	25
3	247.55	10.03	24	246.33	10.49	25	246.45	9.28	21	246.08	8.17	25
5	253.22	10.42	24	252.37	10.22	25	251.79	9.47	21	251.08	9.06	25
8	259.48	10.98	24	259.21	11.18	25	258.14	9.91	21	255.46	8.94	25
11	271.94	11.25	24	271.46	11.31	25	268.92	10.41	21	265.73	10.11	25
14	284.36	11.38	24	284.87	12.15	25	281.01	10.72	21	276.54	10.94	25
17	308.40	12.67	24	307.61	13.93	25	302.82	13.39	21	296.08 *€	16.54	25
20	334.20	12.27	24	335.38	14.88	25	329.56	14.82	21	320.06**€	20.76	25
20^thday Corrected Body Weight (g)	271.44	11.85	24	272.37	12.65	25	270.70	12.48	21	261.48 *€	14.74	25

Key:  N = Number of observations

         SD = Standard deviation

         *= significantly lower than control (p<0.05)

         **= significantly lower than control (p<0.01)

         € =Dunnett's test with Bonferroni adjustment

Note: Values of non-pregnant female rats were not used for statistical analysis.

 Maternal Body Weight Change (%)

 

Dose level	0 mg/kg bw/day			100 mg/kg bw/day			500 mg/kg bw/day			1000 mg/kg bw/day
Gestation Period	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
0-3	3.70	1.11	24	2.94	1.38	25	3.25	1.45	21	3.61	1.70	25
3-5	2.29	1.13	24	2.47	0.93	25	2.17	0.64	21	2.03	1.07	25
5-8	2.47	1.06	24	2.70	1.12	25	2.52	0.81	21	1.75	1.08	25
8-11	4.81	1.27	24	4.74	1.33	25	4.18	1.00	21	4.02	1.17	25
11-14	4.58	1.18	24	4.94	1.01	25	4.50	1.00	21	4.07	1.17	25
14-17	8.46	1.22	24	7.98	1.18	25	7.75	1.64	21	7.03	3.24	25
17-20	8.40	2.10	24	9.05	2.16	25	8.84	1.81	21	8.07	2.78	25
5-20	32.04	3.16	24	32.92	3.58	25	30.91	4.06	21	27.39**€	5.18	25

Key:  N = Number of observations

         SD = Standard deviation

**= significantly lower than control (p<0.01)

         € =Dunnett's test with Bonferroni adjustment

Note: Values of non-pregnant female rats were not used for statistical analysis.

Food Consumption (g/day)

 

Dose level	0 mg/kg bw/day			100 mg/kg bw/day			500 mg/kg bw/day			1000 mg/kg bw/day
Gestation Period	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
0-3	18.71	1.94	24	18.44	2.13	25	18.08	2.53	21	18.89	2.34	25
3-5	20.88	2.71	24	20.92	2.68	25	20.36	2.37	21	21.24	2.09	25
5-8	20.68	2.27	24	20.78	2.75	25	20.20	2.32	21	18.93 *€	2.53	25
8-11	21.28	2.30	24	21.86	1.69	25	20.34	2.73	21	19.75	2.15	25
11-14	23.56	1.46	24	24.34	2.45	25	22.48	2.24	21	21.98 *€	2.20	25
14-17	23.50	2.23	24	23.97	2.69	25	22.11	2.02	21	21.87	2.99	25
17-20	22.48	2.30	24	23.60	3.28	25	21.99	2.73	21	21.48	3.09	25
5-20	22.30	1.02	24	22.90	1.88	25	21.42	1.80	21	20.80 **€	1.80	25

Key:    N = Number of observations

           SD = Standard deviation

           *= significantly lower than control (p<0.05)

           **= significantly lower than control (p<0.01)

           € =Dunnett's test with Bonferroni adjustment

Note:   Values of non-pregnant female rats were not used for statistical analysis.

Prenatal Data

 

Dose level	0 mg/kg bw/day			100 mg/kg bw/day			500 mg/kg bw/day			1000 mg/kg bw/day
Parameter	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
Absolute Uterus Weight (g)	62.75	8.52	24	63.00	5.75	25	58.85	13.60	21	58.57	13.50	25
Relative Uterus Weight (%)	18.77	2.41	24	18.78	1.44	25	17.78	3.75	21	18.18	3.73	25
N^oof CL	13.50	1.44	24	13.12	1.56	25	13.38	1.40	21	13.28	1.86	25
N° of Implants	11.96	1.76	24	12.00	1.04	25	11.57	2.71	21	11.64	2.71	25
N^oof Live Foetus	11.92	1.72	24	11.84	0.99	25	11.19	2.93	21	11.40	2.84	25
N^oof Dead Foetus	0.00	0.00	24	0.00	0.00	25	0.00	0.00	21	0.00	0.00	25
N° of Early Resorption	0.04	0.20	24	0.12	0.33	25	0.24	0.54	21	0.16	0.37	25
N° of Late Resorption	0.00	0.00	24	0.04	0.20	25	0.14	0.48	21	0.08	0.28	25
N° of Total Resorption	0.04	0.20	24	0.16	0.47	25	0.38	0.92	21	0.24	0.52	25
N° of Pre-Implant Loss	1.54	1.67	24	1.12	1.45	25	1.81	2.23	21	1.64	2.18	25
N° of Post Implant Loss	0.04	0.20	24	0.16	0.47	25	0.38	0.92	21	0.24	0.52	25
Pre-Implant Loss (%)	11.06	11.97	24	7.73	9.62	25	13.83	18.77	21	12.47	16.18	25
Post Implant Loss (%)	0.30	1.46	24	1.24	3.58	25	3.53	8.78	21	2.79	8.24	25
Live Foetus (%)	99.70	1.46	24	98.76	3.58	25	96.47	8.78	21	97.21	8.24	25
Dead Foetus (% )	0.00	0.00	24	0.00	0.00	25	0.00	0.00	21	0.00	0.00	25
Total Resorption (%)	0.30	1.46	24	1.24	3.58	25	3.53	8.78	21	2.79	8.24	25

Key: N = Number of observations

        SD = Standard deviation

        CL = Corpora lutea

Note:Values of non-pregnant female rats were not used for statistical analysis

Foetal Data

 

Dose level	0 mg/kg b. wt./day			100 mg/kg b. wt./day			500 mg/kg b. wt./day			1000 mg/kg b. wt./day
Parameter	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
N° of Male Foetus	5.83	2.18	24	5.80	2.10	25	5.52	2.64	21	5.44	1.92	25
N° of Female Foetus	6.08	2.41	24	6.04	1.84	25	5.67	2.61	21	6.21	1.93	24
Total Foetus (Male + Female)	11.92	1.72	24	11.84	0.99	25	11.19	2.93	21	11.40	2.84	25
Male Foetus Weight (g)	3.52	0.21	24	3.54	0.23	25	3.46	0.24	21	3.41	0.37	25
Female Foetus Weight (g)	3.32	0.22	24	3.31	0.20	25	3.28	0.25	21	3.25	0.37	24
Total Foetus Weight (Male + Female)	3.42	0.21	24	3.43	0.20	25	3.36	0.24	21	3.33	0.36	25
AGD – Male	1.80	0.02	24	1.80	0.02	25	1.81	0.02	21	1.83*Φ	0.05	25
AGD – Female	0.89	0.01	24	0.89	0.03	25	0.89	0.01	21	0.90	0.02	24
Male Sex Ratio (%)	49.43	18.30	24	48.63	16.23	25	49.59	19.14	21	49.54	17.59	25

Key: N = Number of observations

        SD = Standard deviation

        M = Male Foetus

        F = Female

        AGD = Anogenital Distance

        * = Significantly higher than control (p<0.05)

        Φ =Cochran's t test

Foetal Gross External Observations

 

Dose level	0 mg/kg bw/day			100 mg/kg bw/day			500 mg/kg bw/day			1000 mg/kg bw/day
N° of Foetuses (Litter) Examined	286 (24)			296 (25)			235 (21)			285 (25)
	A	B	C	A	B	C	A	B	C	A	B	C
Observation:
Abnormality detected	0	0	0.00	0	0	0.00	0	0	0.00	0	0	0.00

Key:   A= Number of foetus affected

          B = Number of litter affected

          C = % Incidence of litter

Foetal Visceral Observations

 

Dose level	0 mg/kg bw/day			100 mg/kg bw/day			500 mg/kg bw/day			1000 mg/kg bw/day
N° of Foetuses (Litters) Examined	135 (24)			144 (25)			115 (21)			137 (25)
	A	B	C	A	B	C	A	B	C	A	B	C
Observation:
Ureter: Dilated	2	2	8.33	0	0	0.00	1	1	4.76	0	0	0.00
Situs inversus	0	0	0.00	0	0	0.00	1	1	4.76	0	0	0.00

Key:    A = Number of foetus affected

B = Number of litter affected

C = % Incidence of litter

Foetal Head Razor Section Observations

 

Dose level	0 mg/kg bw/day			100 mg/kg bw/day			500 mg/kg bw/day			1000 mg/kg bw/day
N° of Foetuses (Litters) Examined	135 (24)			144 (25)			115 (21)			137 (25)
	A	B	C	A	B	C	A	B	C	A	B	C
Observation:
Abnormality detected	0	0	0.00	0	0	0.00	0	0	0.00	0	0	0.00

Key:    A= Number of foetus affected

B = Number of litter affected

C = % Incidence of litter

 Foetal Skeletal Observations

 

Dose level	0 mg/kg bw/day						100 mg/kg bw/day					500 mg/kg bw/day					1000 mg/kg bw/day
N° of Foetuses (Litters) Examined	151 (24)						152 (25)					120 (21)					148 (25)
	A		B		C	A		B		C		A		B		C		A		B		C
Observation^$:
11^thThoracic centrum:Dumbbell ossification	2	2		8.33			0		0		0.00	0	0		0.00		0		0		0.00
14^thRib:Extra ossification centre	26	14		58.33			9#*		9		36.00	18	8		38.10		13#*		9		36.00
1^stSternebrae:Unossified	0	0		0.00			0		0		0.00	2	1		4.76		0		0		0.00
2^ndSternebrae:Incomplete ossification	0	0		0.00			0		0		0.00	1	1		4.76		0		0		0.00
2^ndSternebrae:Unossified	0	0		0.00			3		2		8.00	7#*	2		9.52		3		3		12.00
3^rdSternebrae:Bipartite ossification	0	0		0.00			0		0		0.00	0	0		0.00		1		1		4.00
3^rdSternebrae:Unossified	0	0		0.00			0		0		0.00	2	1		4.76		0		0		0.00
3^rdSternebrae:Misshapen	1	1		4.17			0		0		0.00	0	0		0.00		1		1		4.00
3^rdSternebrae:	0	0		0.00			0		0		0.00	0	0		0.00		1		1		4.00
4^thSternebrae:Bipartite ossification	0	0		0.00			0		0		0.00	0	0		0.00		1		1		4.00
4^thSternebrae:Unossified	0	0		0.00			0		0		0.00	2	1		4.76		0		0		0.00
4^thSternebrae:Misshapen	1	1		4.17			1		1		4.00	0	0		0.00		2		2		8.00
5^thSternebrae:Dumbbell ossification	1	1		4.17			1		1		4.00	1	1		4.76		1		1		4.00
5^thSternebrae:Incomplete ossification	7	6		25.00			3		3		12.00	2	2		9.52		1		1		4.00
5^thSternebrae:Unossified	14	9		37.50			10		7		28.00	10	5		23.81		13		10		40.00
Caudal centrum:Unossified	0	0		0.00			0		0		0.00	2	1		4.76		0		0		0.00
Interparietal:Incomplete ossification	7	4		16.67			3		2		8.00	5	4		19.05		4		4		16.00
Occipital:Bipartite ossification	0	0		0.00			1		1		4.00	0	0		0.00		0		0		0.00
Occipital:Incomplete ossification	1	1		4.17			0		0		0.00	3	2		9.52		0		0		0.00
Parietal: Incomplete ossification	2	2		8.33			1		1		4.00	2	1		4.76		0		0		0.00
Pubic: Unossified	0	0		0.00			0		0		0.00	2	1		4.76		0		0		0.00
Rib: Full supernumerary	2	2		8.33			1		1		4.00	3	3		14.29		1		1		4.00
Rib: Short supernumerary	12	8		33.33			11		7		28.00	12	10		47.62		12		8		32.00
Sacral centrum: Unossified	0	0		0.00			0		0		0.00	1	1		4.76		0		0		0.00
Sacral vertebra: Unossified	0	0		0.00			0		0		0.00	1	1		4.76		0		0		0.00
Squamosal: Incomplete ossification	1	1		4.17			0		0		0.00	2	1		4.76		0		0		0.00
Xiphisternum: Incomplete ossification	12	5		20.83			11		7		28.00	14	9		42.86		17		9		36.00
Xiphisternum: Unossified	1	1		4.17			2		2		8.00	6#*	4		19.05		18#*		11#*		44.00

Key:   $ = One Foetus may have more than one Observation

          * = Chi-square test

A = Number of foetus affected

B = Number of litter affected

C = % Incidence of litter

#= Significantly different than control (p<0.05)

Terminal Body Weight and Thyroid Weight

Parameters	Treament
	0 mg/kg bw/day			100 mg/kg bw/day			500 mg/kg bw/day			1000 mg/kg bw/day
	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
B. wt. -TS (g)	334.2000	12.2736	24	335.3760	14.8779	25	329.5571	14.8163	21	320.0600**€	20.7559	25
Absolute Organ Weight
Thyroid gland (g)	0.0150	0.0025	24	0.0149	0.0026	25	0.0156	0.0023	21	0.0145	0.0023	25
Relative Organ Weight
Thyroid gland (%)	0.0045	0.0007	24	0.0044	0.0007	25	0.0047	0.0007	21	0.0045	0.0008	25

Key: € =Dunnett’s t test with Bonferroni adjustment

        **= 1%

Gross and Microscopic Findings

Treatment		0 mg/kg bw/day	100 mg/kg bw/day	500 mg/kg bw/day	1000 mg/kg bw/day
Organs & Lesions	Sex	F	F	F	F
	N° of Animals	25	25	25	25
GROSS FINDINGS
External
No abnormality detected		25	25	25	25
Internal
No abnormality detected		25	25	25	25
MICROSCOPIC FINDINGS		N° of Dams for Summary of Microscopic Finding
		24*	25	25	25
Thyroid glands
Ultimobranchial cyst		1	X	X	0

Key:  ‘X’ = Organs not examined

          * = Non pregnant dam was not considered for summary of histopathological evaluation.

 

 

Thyroid hormone analysis

Treatment	Mean	SD	N
TSH (ng/mL)
0mg/kg bw/day	0.862	0.274	24
100mg/kg bw/day	0.814	0.299	25
500mg/kg bw/day	1.021	0.421	21
1000mg/kg bw/day	0.828	0.265	25
T3 (ng/L)
0mg/kg bw/day	0.240	0.059	24
100mg/kg bw/day	0.206*Φ	0.036	25
500mg/kg bw/day	0.233	0.031	21
1000mg/kg bw/day	0.232	0.030	25
T4 (ng/L)
0mg/kg bw/day	27.626	7.794	24
100mg/kg bw/day	22.347**Φ	4.108	25
500mg/kg bw/day	24.271	3.925	21
1000mg/kg bw/day	22.834*Φ	4.771	25

Key:    N = Number of observations

           SD = Standard deviation

           Φ=Cochran's t test

           **= Significantly lower than control (p<0.01)

* = Significantly lower than control (p<0.05)

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect Level (NOAEL) is 500 mg/kg bw/day for maternal toxicity and 1000 mg/kg bw/day for foetal toxicity. The LOAEL for maternal toxicity was 1000 mg/kg/day based on adverse effects observed on body weight, body weight change, and food consumption.

Executive summary:

Potential developmental toxicity has been investigated in the rat in a GLP compliant study conducted according to OECD TG 414. The No Observed Adverse Effect Level (NOAEL) was 500 mg/kg bw/day for maternal toxicity and 1000 mg/kg bw/day for foetal toxicity. The LOAEL for maternal toxicity was 1000 mg/kg/day based on adverse effects observed on body weight, body weight change, and food consumption.