Fatty acids, coco, esters with 1,3-butanediol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Oct 2006 - 09 May 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

No analytical purity stated, lack of historical control data.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Deviations:

yes

Remarks:

no analytical purity stated, lack of historical control data.

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

853947-59-8

Cas Number:

853947-59-8

IUPAC Name:

853947-59-8

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Hsd: Sprague Dawley SD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Italy S.r.l. (San Pietro al Natisone (UD), Italy)
- Age at study initiation: 9-10 weeks (females) and 11 weeks (males)
- Weight at study initiation: 202-223 g (females) and 335-342 g (males)
- Housing: Before and after pairing, the animals were housed in groups of no more than 5 per sex in clear polycarbonate cages measuring 59x38.5x20 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Varese, Italy).
- Diet: laboratory rodent diet (4 RF 21, Mucedola S.r.l., Settimo Milanese, Italy), ad libitum
- Water: drinking water, ad libitum
- Acclimation period: approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The required amount of the test item was suspended in the vehicle (corn oil) at a concentration of 500 mg/mL. The formulations were prepared daily and concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Concentration in vehicle: 500 mg/mL
- Amount of vehicle (if gavage): 2 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed to confirm that the proposed formulation procedure was acceptable. The stability of the formulations was found to be 24 h at room temperature. Samples of the formulations prepared during the first and last week of the study were analysed to check the concentration and homogeneity. Homogeneity and recovery were within the acceptable limits.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Proof of pregnancy: sperm in vaginal smear or the presence of a copulation plug was referred to as Day 0 of pregnancy (Day 0 post coitum)
- Other: After proof of pregnancy, the female was separated from the male, which was paired with a new stock female. One male was used to mate with no more than three females.

Duration of treatment / exposure:

Day 6-19 post mating

Frequency of treatment:

daily, 7 days/week

Duration of test:

Day 20 post mating

Doses / concentrations

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24 P females

Control animals:

yes, concurrent vehicle

Details on study design:

- Route selection rationale: The oral route of administration was selected because it is a possible route of exposure of the test item in man.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, prior to dosing, approximately 30 min and 2 h after dosing

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0, 6, 9, 12, 15 and 20 post coitum

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: a detailed post mortem examination was conducted (including examination of the external surface and orifices, no further details)

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:
- gross evaluation of placentae
- Number and distribution of intra-uterine deaths

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Other: number, sex and weight of all live foetuses; number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing)

Statistics:

For continuous variables (body weight, body weight gain, food consumption), the significance of the difference amongst group means was assessed by analysis of variance. Differences between the treated group and the control group were assessed by Dunnett’s test using a pooled error of variance. The homogeneity of the data was assessed by Bartlett’s test before Dunnett’s test was performed. If the data were found to be inhomogeneous, a modified t-test (Cochran and Cox) was applied. The criteria for statistical significance were p < 0.05 and 0.01. The non-parametric Kruskal-Wallis analysis of variance was used for all other parameters (gravid uterus weight, corrected body weight, litter data and sex ratio). Intergroup differences between the control and the treated group were assessed by the non-parametric version of the Williams' test using the SAS System Program. The criteria for statistical significance was p < 0.05. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Indices:

Pre-implantation loss was calculated as a percentage from the formula: [(no. of corpora lutea - no. of implantations) / no. of corpora lutea] x 100

Post-implantation loss was calculated as a percentage from the formula: [(no. of implantations - no. of live young) / no. of implantations] x 100

Total implantation loss was calculated as a percentage from the formula: [(no. of corpora lutea - no. of live young) / no. of corpora lutea] x 100

Sex ratios of the foetuses were calculated as the percentage of males per litter.

All derived values (e.g., means, percentages, ratios) were first calculated within the litter and the group values derived as a mean of individual litter values. Foetal structural deviations were expressed as the percentage of affected foetuses relative to all foetuses examined per group, as well as in terms of the mean litter percentage of affected litters.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No mortality occurred during the study. One treated female was found not pregnant at necropsy. The numbers of dams with live foetuses at necropsy were 24 in the control group and 23 in the treated group. No reactions to treatment were noted at pre- and post-dose observations. Animals did not show any treatment-related effects during clinical sign observations. Body weight, body weight gain and food consumption were unaffected by treatment. No treatment-related effects were seen in terminal body weight, uterus weight or absolute weight gain. No macroscopic changes were detected in treated females.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: overall effects

2

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: overall effects

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
A slight but statistically significant lower mean foetal weight was observed in the treatment group when compared to the control group. This slight difference was attributed to the higher presence of foetuses in the treated group compared to controls in which mean foetal weight was also unusually higher when compared to the internal historical control data. The mean foetal weight of the treated group was well within the historical control data for this rat species (3.03 - 4.2 g, mean 3.6 g). Three small foetuses were present at necropsy: one in the control group and 2 in the treated group. One control foetus showed multiple anomalies: short forelimb, short hindlimb, flexed paw, dome shape of head and short body. Short hindlimb was also observed in one foetus of the treated group. All changes were considered incidental. Slight changes in the skeletal examination of the foetuses were noted between the treated and the control group and were considered spontaneous in origin and not related to treatment. Visceral examination of foetuses showed unilateral cryptorchidism (ectopic testis positioned just below the kidney) in 2 foetuses from 2 different litters (Dam numbers 55 and 75) of the treated group. The identification of the same stock male being mated with these two females (male no. 4) gave rise to the hypothesis that the male parent could genetically transmit the finding. In addition, considering also the high presence of displaced testes in the control group, the findings described were considered evidence of spontaneous pathology, often seen in this species under our experimental conditions.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Table 1. Body weight of pregnant females (g) as group mean data.

Group	Day of gestation phase
		0	6	9	12	15	20
Control	n	24	24	24	24	24	24
	Mean	243.06	272.67	285.87	302.99	324.65	408.35
	SD	14.42	13.62	13.24	14.33	14.67	22.33
Treatment group	n	23	23	23	23	23	23
	Mean	240.36	269.66	282.13	299.15	322.08	408.67
	SD	13.94	12.77	12.47	14.1	16.75	22.36

Table 2. Litter data and sex ratios as group mean data.

Group		Corpora Lutea	Implantations	Uterine Deaths			Viable Young. Total	% Males	Implantation loss			Litter weight (g)	Mean foetal weight (g)
				Early	Late	Total			Pre	Post	Total
Control	n	24	24	24	24	24	24	24	24	24	24	24	24
	Mean	16.58	15.0	0.42	0.21	0.63	14.88	52.93	7.01	3.80	10.36	55.57	3.75
	SD	2.38	2.72	0.65	0.83	0.97	2.61	13.05	8.82	5.84	9.69	10.40	0.37
Treatment group	n	23	23	23	23	23	23	23	23	23	23	23	23
	Mean	17.83	17.17	0.52	0.52	1.04	16.13	50.75	3.77	5.99	9.45	57.97	3.59*
	SD	1.59	2.04	0.79	2.09	2.12	2.78	8.02	5.90	12.33	13.40	10.33	0.19

* = mean value of group is significantly different from controls at p < 0.05 (William’s test)

Table 3. Litter results.

	Control	Treatment group
Anomalies
Small foetus	1	2
Short forelimb, flexed paw, dome shape of head and short body	1	0
Short hindlimb	1	1
Visceral examination
Unilateral cryptorchidism	0	2

Applicant's summary and conclusion

Conclusions:

The test substance had no effect on intrauterine development.