Morpholine, 4-C12-14-alkyl derivs.

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented and cited in the peer-reviewed regulatory agency.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-ethylmorpholine

EC Number:

202-885-0

EC Name:

4-ethylmorpholine

Cas Number:

100-74-3

IUPAC Name:

4-ethylmorpholine

Constituent 2

Reference substance name:

Morpholine, 4-ethyl-

IUPAC Name:

Morpholine, 4-ethyl-

Details on test material:

- Name of test material (as cited in study report): N-ethylmorpholine
- Analytical purity: Equal or more than 99 %
- Impurities (identity and concentrations): 0.05% as moisture
- Lot/batch No.: 2901P0

Test animals

Species:

rat

Strain:

other: Crj:CD(SD)IGS

Sex:

male/female

Details on test animals or test system and environmental conditions:

Four weeks old Crj:CD(SD)IGS rats bought from Charles River Japan, Inc. They were put in quarantine and acclimatization for 7 d before use.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test solution was prepared and diluted to dosing concentrations by injection solvent every week. They were kept in a refrigerator. The diluted solution was also confirmed to be stable for 8 d.

Duration of treatment / exposure:

Males: 42 d
Females: From 14 d before mating to Day 3 of lactation

Frequency of treatment:

Once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (vehicle), 50, 150, 500 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

13/sex/dose

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

Oestrous cyclicity (parental animals):

Yes

Sperm parameters (parental animals):

Parameters examined in P male parental generations: Testis weight, epididymis weight, histopathological examination of reproductive organs

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Number and sex of pups, stillbirths, live births, viability index

Postmortem examinations (parental animals):

Terminal killing: Males at Day 43; females at Day 4 of lactation.

Postmortem examinations (offspring):

Necropsy was perfomed.

Statistics:

Statistical analysis of offspring was carried out using the litter as the experimental unit. Bartlett's test of homogeneity of variance was used to determine if the groups had equivalent at the 5% level of significance. If variance was equivalent, the groups were compared by one-way analysis of variance. If significant
differences were found, Dunnett's test was performed. If the groups did not have equivalent variances, the Kruskal-Wallis test was used to assess the overall effects. Whenever significant differences were noted, Dunnett-type test was performed. Mann-Whitney U-test or Fisher's exact test was also used.

Reproductive indices:

Copulation index and fertility index

Offspring viability indices:

Gestation index , implantation index, delivery index , birth index , live birth index , sex ratio and viability index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

No deaths were found in males of any group. One female at 500 mg/kg bw/day showed tremor and died on Day 2 of lactation. In clinical signs, salivation was observed in males and females at 150 mg/kg bw/day and higher. Decrease in body weight gain and food consumption were detected in male at 500 mg/kg bw/day and females at 150 mg/kg bw/day and higher. Absolute and relative weights of testes and epididymides were not affected by dosing. Necropsy and histopathological examination of reproductive organs revealed no abnormalities related to dosing. No adverse effects on estrous cycle, copulation, fertility, gestation length, gestation index or number of corpora lutea were found. No significant changes were observed in numbers of implantations, pups live pups and in indexes for implantation, delivery, birth and live birth were detected.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

(for reproductive toxicity)

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Remarks:

(for systemic toxicity)

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on observed clinical signs such as salivation as well as decrease in body weight and food consumption at higher doses.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Gross pathological findings:

no effects observed

Details on results (F1)

There were no treatment-related changes in the body weight external appearance, general conditions or necropsy findings in offspring of rats.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1. Body weight changes of rats treated orally with N-ethylmorpholine in preliminary reproduction toxicity screening test:

Dose (mg/kg/ bw/day)	0	50	150	500
Male No. of animals	13	13	13	13
Terminal body weight (g) +/-S.D.	497.0     40.4	494.0     30.9	488.0     38.9	457.5* 31.7
Female No. of animals	13	13	13	13
Days 14 of administration (g) +/-S.D.	266.5     13.1	263.6     15.7	263.4     17.8	259.5 17.5
No. of animals	12	12	12	12
Days 20 of pregnancy (g) +/-S.D.	428.5     33.1	423.2     29.8	410.6     27.2	386.1*   42.1
No. of animals	12	12	12	12
Days 4 of lactation (g) +/-S.D.	345.6     22.8	338.4 26.9	330.1 19.3	329.7 24.5

Significant difference from control group; *p=<0.05,**p=<0.01

Table 2. Food consumption of rats treated orally with N-ethylmorpholine in preliminary reproduction toxicity screening test

Dose (mg/kg/ bw/day)	0	50	150	500
Male No. of animals	13	13	13	13
Days of administration (g)
1-2 +/-S.D.	27.8        1.9	26.9 2.3	25.4 3.7	21.3** 2.7
7-8 +/-S.D.	25.1 1.9	25.8 1.5	25.3 2.8	22.1** 1.7
14-15     +/-S.D.	27.1 2.9	27.7 8.2	26.6 2.9	24.3* 3.2
29-30 +/-S.D.	28.7 2.4	29.5 2.1	28.0 2.7	24.2** 1.7
35-36 +/-S.D.	29.9 7.5	26.7 3.3	27.7 3.0	27.2 6.4
41-42     +/-S.D.	29.6        3.3	29.4 3.4	28.9 4.1	26.2* 2.0
Female No. of animals	13	13	13	13
Days of administration (g)
1-2 +/-S.D.	20.7 1.9	18.5 3.5	16.6** 3.4	16.5** 3.7
7-8          +/-S.D.	19.7 3.7	19.4 3.9	20.2 2.9	19.9 3.5
14-15 +/-S.D.	19.9 3.7	20.4 2.6	19.7 2.3	19.4 1.8
No. of animals	12	12	12	12
Days of pregnancy (g)
0-1 +/-S.D.	21.0        2.6	21.1 1.9	19.4 2.7	18.4* 2.9
7-8          +/-S.D.	28.9 3.6	28.5 3.0	25.5* 3.4	23.2** 3.2
14-15     +/-S.D.	28.6 3.4	26.8 2.8	26.3 2.0	24.7* 4.3
20-21     +/-S.D.	19.3 3.5	21.0 2.4	20.8 4.0	18.7 2.5
No. of animals	12	12	12	9
Days 4 of lactation (g)
3-4 +/-S.D.	47.1 6.7	49.4 4.6	43.3 4.3	41.4        8.8

Significant difference from control group; *p=<0.05,**p=<0.01

Table 3. Estrous cycle and reproductive performance in rats treated orally with N-ethylmorpholine in preliminary reproduction toxicity screening test

Dose (mg/kg/ bw/day)	0		50	150	500
Estrous cycle
Type of cycle during treatment period
4-day cycle		13	13	12	12
4,5-day cycle		0	0	1	1
Length of estrous cycle in days +/-S.D.		4.0 0.0	4.0 0.0	4.0 0.1	4.0 0.1
Reproductive performance
Number of mated pairs		13	13	13	13
Number of copulated pairs		13	13	12	12
Copulation index (%)		100.0	100.0	92.3	92.3
Number of pregnant animals		12	12	12	11
Fertility index (%)		92.3	92.3	100.0	91.7
Pairing days until copulation  +/-S.D.		2.3 1.1	2.2 1.1	3.3 1.7	2.6 1.3
Frequency of vaginal estrus  +/-S.D.		1.0 0.0	1.0 0.0	1.1 0.3	1.0 0.0

Copulation index = (Number of copulated pairs/Number of mated pairs) X 100
Fertility index = (Number of pregnant animals/Number of copulated pairs) X 100

Table 4. Summary of development of pups from dams treated orally with N-ethylmorpholine in preliminary reproduction toxicity screening test

Dose (mg/kg/ bw/day)	0	50	150	500
Number of pregnant females	12	12	12	11
Gestation index (%)	100.0	100.0	100.0	90.9
Gestation length in days +/-S.D.	22.7 0.5	22.3 0.7	22.3 0.5	22.8 0.6
Number of corpora lutea +/-S.D.	16.2 2.0	16.9 1.8	15.7 1.9	16.0 3.0
Number of implantation sites +/-S.D.	15.6 2.6	16.1 1.7	14.8 1.8	13.1 4.7
Implantation index (%) +/-S.D.	95.9 7.4	95.2 4.9	94.9 6.1	80.6 26.6
Day 0 of lactation
Number of pups born +/-S.D.	14.2        2.8	15.1 1.2	13.8 1.6	11.4 4.9
Delivery index (%) +/-S.D.	90.6 9.3	94.1 6.0	93.1 7.7	84.6 15.8
Number of pups alive +/-S.D.	13.7 2.9	15.0 1.3	13.7 1.7	10.0 5.4
Birth index (%) +/-S.D.	87.4 9.7	93.6 5.7	92.5 8.0	71.2 32.7
Live birth index (%) +/-S.D.	96.5 5.6	99.4 1.9	99.4 2.2	83.5 35.6
Sex ratio on day 0 (%) +/-S.D.	52.4 13.2	47.9 8.0	48.9 12.6	54.2 26.3
Day 4 of lactation
Number of pups alive +/-S.D.	13.6 2.8	15.0 1.3	13.5 1.7	11.6 3.9
Viability index (%) +/-S.D.	99.5 1.7	100.0 0.0	98.8 2.8	98.1 3.8
Sex ratio on day 4 (%) +/-S.D.	52.6 13.1	47.9 8.0	49.5 12.6	58.5 25.9

Gestation index = (Number of females with live pups/ Number of pregnant females) X 100
Implantation index = (Number of implantation sites/ Number of corpora lutea) X 100
Delivery index = (Number of pups born/Number of implantation sites) X 100
Birth index = (Number of live pups on day 0/Number of implantation sites) X 100
Live birth index = (Number of live pups on day 0/Number of pups born) X 100
Sex ratio = (Number of male live pups/Number of female live pups) X 100
Viability index = (Number of live pups on day 4/Number of live pups on day 0)

Applicant's summary and conclusion

Conclusions:

Based on the absence of any adverse effects of the read-across substance, ethylmorpholine on reproductive and developmental parameter, the NOAEL for reproductive and developmental toxicity is considered to be 500 mg/kg bw/day. Further, based on clinical signs and decreased body weight gain and food consumption, the LOAEL and NOAEL for general toxicity in parent animals are considered to be 150 and 50 mg/kg bw/day, respectively.

Executive summary:

The toxicity to reproduction of the read-across substance, 4-ethylmorpholine was tested in a reproduction / developmental toxicity screening test according to the OECD Guideline 421 in compliance with GLP.

Drj:CD(SD)IGD rats (13 animals/sex/dose) were given test substance by gavage at 0 (vehicle: water), 50, 150 or 500 mg/kg bw/day. Males were dosed for a total of 42 d beginning 14 d before mating. Females were dosed from 14 d before mating to Day 3 of lactation throughout the mating and pregnancy period.

No deaths were found in males of any group. One female at 500 mg/kg bw/day died on Day 2 of lactation. Tremor was observed in the female which died, and transient salivation after dosing was observed in males and females at 150 mg/kg bw/day and higher. Decrease in body weight gain accompanied by reduced food consumption was detected in males at 500 mg/kg bw/day and females at 150 mg/kg bw/day and higher. Body weight gains on Days 1-7, Days 21 -28 and Days 35-43 of administration in males at 500 mg/kg bw/day, on Days 1-7 of administration, Days 7-14 and Days 14-21 of pregnancy and Days 0-4 of lactation in females at 500 mg/kg bw/day, and Days 1-7 of administration, Days 0-7 of pregnancy and Days 0-4 of lactation in females at 150 mg/kg bw/day were found. Absolute and relative weights of the testes and epididymides in the groups treated with this chemical were not different from the control group. Necropsy and histopathological examinations revealed no changes related to the administration of this chemical. Histopathological examinations of the testes, epididymides and ovaries revealed no toxicological changes. There were no adverse effects on estrous cyclicity, copulation index, fertility index, precoital interval, gestation length, gestation index or number of corpora lutea. No significant changes were observed in numbers of implantations and pups and live pups, and in indexes for implantation, delivery, birth and live birth. There were no treatment- related changes in body weight, external appearance or necropsy findings in offspring of rats.

Based on the absence of any adverse effects on reproductive and developmental parameter, the NOAEL for reproductive and developmental toxicity is considered to be 500 mg/kg bw/day. Further, based on clinical signs and decreased body weight gain and food consumption, the LOAEL and NOAEL for general toxicity in parent animals are considered to be 150 and 50 mg/kg bw/day, respectively.