A mixture of branched and linear C7-C9 alkyl 3-[3-(2H-benzotriazol-2-yl)-5-(1,1-dimethylethyl)-4-hydroxyphenyl]propionates

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP and OECD guideline compliant study with well characterized test material. A reduction in reliability is assigned for use as read-across.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of: α-3-(3-(2H-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl)propionyl-ω-hydroxypoly(oxyethylene); α-3-(3-(2H-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl)propionyl-ω-3-(3-(2H-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl)propionyloxypoly(oxyethylene)

EC Number:

400-830-7

EC Name:

A mixture of: α-3-(3-(2H-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl)propionyl-ω-hydroxypoly(oxyethylene); α-3-(3-(2H-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl)propionyl-ω-3-(3-(2H-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl)propionyloxypoly(oxyethylene)

Molecular formula:

The substance is a mixture of a number of chemical entities, of which there are two principal components : (C2H4O)6-7C19H21N3O3 (major product) (C2H4O)6-7C38H40N6O5 (minor product)

IUPAC Name:

400-830-7

Test material form:

liquid: viscous

Details on test material:

- Physical state: liquid (clear yellow)
- Analytical purity: 100 % (UVCB substance)
- Expiration date of the lot/batch: 15. October 2015
- Stability under test conditions: stable
- Storage condition of test material: sealed container, 15 -25°C, in the dark
- Other: alternative identifiyer: EC 400-830-7
- Structural formula attached as image file (if other than submission substance): see Fig. for the teratogenicity study in rats.

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Covance Research Products, Pennsylvania, USA.
- Age at study initiation: no data
- Weight at study initiation: At mating the female rabbits weighed at least 2.5 kg
- Fasting period before study: none
- Housing: single cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: three days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 to 21°C
- Humidity (%): at least 45%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 30. April 2013 To: 23 May 2013

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous carboxymethylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations of the test article in the vehicle were prepared daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Substance is not soluble in water, but dispersible in the vehicle.
- Concentration in vehicle: adjusted to dose
- Amount of vehicle (if gavage): 10 mL/kg.

All animals were given a small amount of irradiated meadow hay (IPS, Finedon, Northants) at intervals throughout the study. This was offered as environmental enrichment only and was not included in any food consumption calculations.
On several occasions during the study it was necessary to provide some animals with hay moistened with water due to inappetance. This was not included in any food consumption calculations.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples from formulations prepared for use on the first day of dosing were taken for analysis of achieved concentration. In addition three samples were taken from formulations on the last day of dosing. The mean of the homogeneity results was taken as the achieved concentration where the sampling occasion coincided.
Samples were analysed by the Formulations Analysis section of the contract research facility.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant (The females were delivered on Day 3 of gestation.)

Duration of treatment / exposure:

Day 6 to Day 28 of gestation, inclusive

Frequency of treatment:

daily

Duration of test:

Animals were sacrificed on gestaton day 29.

Doses / concentrations

Remarks:

Doses / Concentrations:
10, 30 and 100 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

Based on the dose-range-finding study in five pregnant rabbits per group, 10 mg/kg bw was expected to show no maternal toxicity. The dose level of 300 mg/kg bw dosed to pregnant rabbits in study 8280620 resulted in body weight loss and animals had to be sacrificed. A dose levels of 150 mg/kg/day elicited reduced food intake and reduced body weight gain. A dose level of 50 mg/kg/day elicited slightly increased liver weight only, and so a dose level of 100 mg/kg/day was deemed to be a suitable high dose level for this study.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: beginning and end (nominal) of the working day.
- Post dosing observation: Daily: upon return to the home cage and at 0.5, 1, 2 and 4 hours post dose.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Days 0 (at supplier), 3, 6, 7, 8, 9, 12, 15, 17, 19, 22, 25, 28 and 29 of gestation.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0 (at supplier), 3, 6, 7, 8, 9, 12, 15, 17, 19, 22, 25, 28 and 29 of gestation.

FOOD CONSUMPTION : Yes
- Food consumption for each animal determined (grams) and calculated as g/animal/day.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29

OTHER:

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: intrauterine position of implantation sites

- numer of live/dead foetuses

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter

external foetal assessment
foetal weight
placental weight
foetal sex

Statistics:

All variables were analysed with a twosided risk except where stated below. Body weight gains, Day 29 corrected body weights, food intake and litter weights were analysed using one-way analysis of variance (ANOVA). Levene's test for equality of variances among the groups was performed. Where this showed no evidence of heterogeneity (P0.01), pairwise comparisons with control were made using Dunnett's test. A linear contrast was performed to determine whether there was a relationship between increasing dose and response. A significant trend (P<0.05) was only reported where none of the pairwise comparisons was significant. Where Levene’s test showed evidence of heterogeneity (P<0.01), as the data were unsuitable for log-transformation, non-parametric methods were used. Placental weights, the percentage of male foetuses and the numbers of corpora lutea, implantations and foetuses per female were analysed using non-parametric methods. The non-parametric methods employed were the Kruskal-Wallis ANOVA, the Terpstra-Jonckheere test for a dose related trend and the Wilcoxon rank sum test for pairwise comparisons. Where the Kruskal-Wallis ANOVA was not significant, the pairwise comparisons were not reported in order to protect the Type I error. Foetal weights were analysed using Analysis of Covariance (ANCOVA) and Dunnett's test, using the litter size as covariate. This analysis depends on the assumption that the relationship between the foetal weight and the covariate is the same for all groups, and the validity of this assumption was tested. Levene's test for equality of variances across the groups was also performed and this showed no evidence of heterogeneity (P<=0.01). Gravid uterus weights were analysed using Analysis of Covariance (ANCOVA) and Dunnett's test, using the corrected body weight on day 29 as covariate. This analysis depends on the assumption that the relationship between the organ weight. Further information available, but maximum size limit reached.

Indices:

Percentage pre-implantation loss
Percentage post-implantation loss
Percentage male foetuses

Historical control data:

included in report for 6 studies performed by supplier

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Food consumption was dose-dependently reduced at 100 and 30 mg/kg bw/day (Table 1, data for females excluding those with aborations). Body weight gains between gestation day 6 and 29 were decreased by approximately 20% at 30 mg/kg bw/day, and by approximately 70% at 100 mg/kg bw/day (Figure 1 and Table 3).
In animals receiving 100 mg/kg/day eight animals were killed after aborting their pregnancies between Days 20 to 28 of gestation (Table 2). Many of these animals had few faeces, poor food consumption and body weight loss, so their demise was considered to be treatment-related. The abortion is considered to be a consequence of the maternal toxicity as indicated by the very poor food consumption.

There were no post-dosing observations and no further clinical signs.
Other than the thin animals, there were no treatment-related findings at necropsy (Table 7).

Individual data on food consumption and body weights are given in the attachment.

Effect levels (maternal animals)

Dose descriptor:

NOEL

Effect level:

10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Mean foetal weight and therefore mean litter weight were reduced compared with controls at 100 mg/kg/day (17% and 25%, respectively, table 5) and this was considered to be as a result of the marked maternal toxicity seen at that dose level. Other foetal parameters were unaffected by treatment (see table 6 and attachment).

Effect levels (fetuses)

Dose descriptor:

NOEL

Effect level:

>= 100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Table 1: Mean food intake of dams (excluding females with abortions)

	Mean food intake (g/animal/day)
Days of gestation	control	10 mg/kg bw	30 mg/kg bw	100 mg/kg bw	Statistics
3-5	157	153	157	160	X
6	159	162	133	112**	A
7	150	146	129	82***	A
8	146	144	112**	64***	A
9-11	141	131	108*	61***	A
12-14	123	100	81*	51***	A
15-16	130	100	87*	58***	A
17-18	152	133	105**	87***	A
19-21	132	117	103	98*	A
22-24	108	108	101	80*	A
25-27	96	96	94	48***	A
28	96	93	95	55***	A
A = ANOVA, dose response and Dunnett's			* P<0.05
X = not analysed			** P<0.01
			*** P<0.001

Table 2 Summary of female performance

	control	10 mg/kg bw	30 mg/kg bw	100 mg/kg bw
In group	24	24	24	24
Not pregnant	1	3	1	0
Pregnant (%)	23 (95.8)	21 (87.5)	23 (95.8)	24 (100.0)
Aborted and killed	0	0	0	8
Died/killed*	2	0	2	1
With total embryo/foetal loss	0	0	0	0
With live foetuses on Day 29	21	21	21	15

*Death was due to dosing errors and not related to treatement.

Table 3: Group mean body weights (kg) excluding females with abortions
day of gestation	control	10 mg/kg bw	30 mg/kg bw	100 mg/kg bw
3	3.24	3.20	3.22	3.22
6	3.35	3.34	3.35	3.34
7	3.35	3.33	3.33	3.31
8	3.36	3.33	3.31	3.26
9	3.36	3.34	3.30	3.25
12	3.41	3.36	3.32	3.24
15	3.47	3.42	3.37	3.30
17	3.50	3.44	3.38	3.34
19	3.53	3.48	3.41	3.37
22	3.60	3.54	3.47	3.40
25	3.64	3.59	3.53	3.45
28	3.67	3.63	3.57	3.44
29	3.69	3.67	3.63	3.45
% body weight change; days
3-6	3.3	4.3	3.9	3.7
6-28	9.7	9.0	6.9	2.9
28-29	0.6	1.1	1.5	0.5

Table 4: Group mean caesarian data – uterine/implantation data

	control	10 mg/kg bw	30 mg/kg bw	100 mg/kg bw	statistics
Number of females with live foetuses on Day 29 gestation	21	21	21	15
Mean number of corpora lutea per female	10.5	10.7	10.2	10.7	J
Mean number of implantations per female	8.9	10.2	9.1	8.7	J
Pre-implantation loss: mean%	14.0	4.6	10.6	17.6
number of dams affected	15	6	10	9	F+
Early intrauterine deaths: mean number	0.2	0.2	0.2	0.3
number of dams affected	4	3	3	4	F+
Late intrauterine deaths:
mean number	0.1	0.3	0.3	0.3
number of dams affected	2	4	4	3	F+
Dead foetuses:
mean number	0.0	0.0	0.0	0.1
number of dams affected	0	0	0	1	F+
Post-implantation loss
mean%	2.9	4.4	4.8	8.1
number of dams affected	5	7	5	7	F+
Mean number of foetuses per female	8.6	9.8	8.6	8.0	J
F+ = Cochran-Armitage and Fisher's Exact (upper tail)
J = Kruskal-Wallis, Terpstra-Jonckheere, Wilcoxon

Table 5: Group mean caesarian data - foetal data

	control	10 mg/kg bw	30 mg/kg bw	100 mg/kg bw	statistics
Number of male foetuses	99	103	91	65
Number of female foetuses	82	102	90	55
Mean % male foetuses	55.6	49.8	51.4	57.1	J
Mean litter weight (g)	338.8	357.0	315.5	255.2***	A
Mean placental weight (g)	4.92	4.83	4.69	4.91	J
Mean foetal weight (g) Adjusted	39.4	38.1	37.0	31.7***	C
Mean foetal weight (g) Unadjusted	(39.6)	(36.9)	(37.3)	(32.7)
Mean foetal weight (g) - males only, Adjusted	40	37.9	37.7	31.9***	C
Mean foetal weight (g) - males only, Undjusted	(40.3)	(36.8)	(38.0)	(32.9)
Mean foetal weight (g) - females only, Adjusted	38.7	38.0	36.1	30.4***	C
Mean foetal weight (g) - females only, Unadjusted	(39)	(37.0)	(36.4)	(31.0)

J = Kruskal-Wallis, Terpstra-Jonckheere, Wilcoxon

A = ANOVA, dose response and Dunnett's

C = ANCOVA and Dunnett’s

* P<0.05, ** P<0.01, *** P<0.001

Table 6: Group mean foetal defect data

	control	10 mg/kg bw	30 mg/kg bw	100 mg/kg bw	statistics
EXTERNAL/VISCERAL DEFECTS
Number of foetuses examined	181	205	181	120
Number of litters examined	21	21	21	15
Number showing malformations	2	4	3	3
Mean % of foetuses examined	1.1	2.0	1.5	2.0
Number of litters affected	2	2	3	2	F+
Number showing variations	82	125	115	85
Mean % of foetuses examined	46.9	61.5	64.6	69.5
Number of litters affected	20	21	21	15	F+
SKELETAL DEFECTS
Number of foetuses examined	181	205	181	120
Number of litters examined	21	21	21	15
Number showing malformations	1	3	3	2
Mean % of foetuses examined	0.6	1.4	1.6	1.5
Number of litters affected	1	3	3	2	F+
Number showing variations	147	163	146	105
Mean % of foetuses examined	80.8	78.2	81.4	87.6
Number of litters affected	21	21	21	15	F+
Total number of foetuses showing malformations	3	7	5	4
% of foetuses examined	1.7	3.4	2.8	3.3
Number of litters affected	3	5	5	3	F+

F+ = Cochran-Armitage and Fisher's Exact (upper tail)

Table 7: Summary of necropsy findings

	control	10 mg/kg bw	30 mg/kg bw	100 mg/kg bw
Number of animals examined (females with live fetuses on day 29)	21	21	21	15
Number of animals with finding:
Not remarkable (%)	17 (81.0)	15 (71.4)	18 (85.7)	12 (80.0)
Animal - Thin		1	2	3
Ear - Lesion		1	1
Heart - Abnormal contents			1
Heart - Abnormal fluid		1	1
Heart - Raised area			1
Liver - Mottled	2	2
Liver - Pale	2	1
Lung - Adhesion			1
Lung - Dark		2
Lung - Dark focus	1	1	1
Lung - Raised area			1
Ovary - Dark focus		1
Thoracic cavity - Clear fluid			1
Thoracic cavity - Mass multiple			1
Thymus - Dark	1

The formulations were to be considered homogeneous if the coefficient of variation (CV) of the results was equal or less than 6.0% and the homogeneity results were within ± 10% of the mean. The results were within these criteria.The target range for the preparation of the formulations was 90 to 110% of nominal. Results were within this range. Test article was not detected in the Group 1 control samples.

Applicant's summary and conclusion

Conclusions:

The substance is not teratogenic or embryotoxic in rabbits.

Executive summary:

Administration to pregnant rabbits at a dose level of 100 mg/kg/day elicited toxicity in the form of reduced food intake, reduced body weight gain, eight aborted pregnancies, and reduced foetal weight.

The dose level of 30 mg/kg/day elicited effects of a slight reduction in food intake and body weight gain. The maternal No-Observed-Adverse-Effect-Level (NOAEL) was 10 mg/kg/day. The No-Observed-Effect-Level (NOEL) for embryo-foetal development was 100 mg/kg/day.