Pyridine, alkyl derivs.

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Remarks:

CiToxLAB Hungary Ltd.

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

- Name of test material: Pyridine, alkyl derivs.

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, from SPF colony
- Age at study initiation: Young adult female rats, nulliparous and non-pregnant, at least approximately 11 weeks old
- Weight at study initiation: Will not exceed ± 20% of the mean weight at onset of treatment
- Housing: Type II and/or III polycarbonate cages
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 October 2015 To: 12 November 2015

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Justification for use and choice of vehicle (if other than water): Polyethylene glycol 400 was selected based on the previous experimental work
- Concentration in vehicle: 0, 10, 33.3, 100 mg/ml
- Amount of vehicle (if gavage): 3.0 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of test formulations for concentration were performed according to a validated analytical method. Top, middle and bottom samples were taken from test item formulations two times during the study, during the first and last week of treatment.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male / 1 - 3 females
- Length of cohabitation: 2 hours/day
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Gestation day (GD) 6 to GD19

Frequency of treatment:

Daily on a 7 days/week basis

Duration of test:

GD0 to GD20

No. of animals per sex per dose:

24 mated females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were set based on available data including the results of a dose range finding study in the pregnant rat, with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At the onset of treatment (GD6) then at least weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of each animal will be recorded with precision of +/- 1 g at least on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20.


FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes; stomach with the GI tract (oesophagus, small intestine including duodenum, ileum and jejunum with Peyer’s patches and large intestine including caecum, colon and rectum). The ovaries and uterus were removed and the pregnancy status ascertained. The uterus including the cervix were weighed and examined for early and late embryonic or foetal deaths and for the number of live foetuses.

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: Placentas were examined macroscopically

Fetal examinations:

- External examinations: Yes, plus an additional examination of the great arteries
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

The statistical evaluation of data was performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) or SAS 9.2 in the case of Provantis 9, by an appropriate statistical method (Bartlett, ANOVA/ANCOVA and Duncan, Kruskal-Wallis and Mann-Whitney U tests, T-test, Wilcoxon test, Chi2). The homogeneity of variance between groups was checked by Bartlett`s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA/ANCOVA) was be made. If the obtained result was significant Duncan’s Multiple Range test or Kruskal-Wallis test was used to assess the significance of inter-group differences. Significant results with inter-group comparisons will be further compared using Kruskal-Wallis, and Mann-Whitney U-tests.
Non pregnant females, females with no implantation, or ≤ 5 implantation sites, and females showing signs of abnormal pathology and/or misdosing will be excluded from statistical analysis; in-life individual data will be reported as applicable.
The limit for growth retarded foetuses was calculated from the average body weight of the vehicle control foetuses. A foetus was considered as growth retarded if the deviation from the mean control values was greater than minus two fold standard deviation of all control foetuses.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Observed effects include reduced food consumption, body weight, body weight gain, and adjusted/corrected body weight gain.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Observed were reduced foetal weights. This effect is most likely attributed to the effects upon the dams. Therefore, it does not represent a primary effect of treatment per se and is not relevant for classification.

There were no direct effects of the test item on external, visceral and/or skeletal development of foetuses in the study; slight retardation of ossification was associated with foetal body weight and maternal toxicity at the High dose group.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: embryotoxicity

2

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: foetotoxicity

3

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: teratogenecity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Table 1: Summary of pregnancy data

	Dose (mg/kg bw/day)
Parameters	0	30	100	300
Number of mated females	24	24	24	24
Number of non-pregnant females	0	0	0	0
Number of females with ≤ 5 implantation sites	0	0	0	0
Number of evaluated females on GD20 (Caesarean section)	24	24	24	24

Table 2: Summary of the intrauterine evaluation

Parameters	Dose (mg/kg bw/day)
	0	30	100	300
Number of evaluated dams	24	24	24	24
Mean number of corpora lutea	12.21	12.33	12.38	11.50
Mean number of implantations	11.42	11.00	11.33	10.67
Preimplantation loss, mean	0.79	1.33	1.04	0.83
Preimplantation loss (%), mean	6.25	10.21	7.79	7.08
Early embryonic loss, mean	0.38	0.50	0.54	0.38
Early embryonic loss (%), mean	3.04	4.17	5.13	3.33
Late embryonic loss, mean	0.25	0.00	0.33	0.67
Late embryonic loss (%), mean	2.38	0.00	3.00	6.08
Dead foetuses, mean	0.04	0.00	0.00	0.00
Postimplantation loss, mean	0.67	0.50	0.88	1.04
Postimplantation loss (%), mean	6.13	4.17	8.17	9.42
Total intrauterine mortality, mean	1.46	1.83	1.92	1.88
Total intrauterine mortality (%), mean	11.58	14.00	15.00	16.29
Viable foetuses, mean	10.75	10.50	10.46	9.63

Note: No statistically significant differences were observed in these examined parameters of any test item treated groups when compared to the negative (vehicle) control.

Bodyweight (g)

No toxicologically significant changes were observed in the mean bodyweights or body weight gain values of the Low or Mid test item treated groups when compared to control. The High (300 mg/kg bw/day) dose group, had significantly lower bodyweight during the treatment period, correlating with the significantly lower bodyweight gain values during the study.

 

Sex: Female day(s) Relative to Start Date		0 mg/kgbw/day	30 mg/kgbw/day	100 mg/kgbw/day	300 mg/kgbw/day
0	Mean	209.3	210.0	210.0	211.1
6	Mean	232.4	232.8	232.4	232.8
12	Mean	253.1	252.2	249.3	234.9
20	Mean	321.2	319.7	321.8	285.0

Gravid uterus weight and corrected body weight were also significantly lower in the High dose group (p < 0.05 .and p < 0.01, respectively)

In the High dose group (300 mg/kg bw/day), the corrected body weight gain and corrected net body weight gain (body weight gain during the treatment period adjusted for the gravid uterine weight) was lower by approx. 60 and 109%, respectively compared to the Control group; these difference were also statistically significant (p < 0.01). There were no effects in the Low or Mid dose groups.

Statistically significant lower foetus weight per litter was observed in the High dose group, and the number of foetuses with retarded body weight was also increased in this group.

No direct developmental toxicity including teratogenicity was observed at any dose level.

Table 3: Examination of viable foetuses

Parameters	Dose (mg/kg bw/day)
	0	30	100	300
Number of examined litters	24	24	24	24
Viable foetuses, mean	10.75	10.50	10.46	9.63
Male foetuses, mean	4.63	5.54	5.67	4.46
Female foetuses, mean	6.13	4.96	4.79	5.17
Total number of foetuses	258	252	251	231
Total number of male foetuses	111	133	136	107
Total number of female foetuses	147	119	115	124
Mean foetal weight / litter (g)	3.551	3.592	3.573	3.277**
Number of foetuses with retarded body weight	8	3	2	32**
Number of affected litters (Runts)	6	2	2	11

**: p< 0.01

Applicant's summary and conclusion

Conclusions:

Maternal toxicity was evident at 300 mg/kg bw/day. Therefore, the NOAEL for maternal toxicity is 100 mg/kg bw/day. The NOAEL/LOAEL for developmental effects was the same as that for maternal/systemic effects and is based on reduced foetal weights. The effect upon foetal weights is most likely attributed to the effects upon the dams. Therefore, it does not represent a primary effect of treatment per se and is not relevant for classification.