Bis(2-hydroxyethyl)ammonium acetate

Toxicity to reproduction: other studies

Administrative data

Endpoint:

toxicity to reproduction: other studies

Type of information:

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

Adequacy of study:

key study

Study period:

2006-11-06 to 2008-01-10

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study is the result of a structural analogue substance used as read-across substance. Study is conducted according to Guidelines in a GLP certified laboratory.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 415 (One-Generation Reproduction Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of method:

in vivo

Test material

Test material information

Constituent 1

Reference substance name:

RA2

IUPAC Name:

RA2

Test material form:

other: solid

Details on test material:

Description: Light Yellow Solid
Stability of test compound: Stable

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

- Males: The test item was administered during a 70-day pre-pairing period, during the pairing period and until necropsy
- Females: The test item was administered during a 14-day pre-pairing period and also during the pairing, gestation and lactation periods until necropsy

Frequency of treatment:

Once daily

Duration of test:

Total duration of the test was 120 days

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (control), 100, 300 and 1000 mg/kg bw/day
Basis:
nominal in water

No. of animals per sex per dose:

24 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The test item was administered during a 70-day pre-pairing period to 24 males per group once daily, in the morning. At 14 days prior to pairing, the test item was administered daily to 24 females per dose group. After the pre-pairing treatment period, parental animals were paired with one male/one female per cage for a maximum of 14 days. Vaginal smears were taken daily throughout the pairing period until either spermatoa or a vaginal plug were observed. This day was designated day 0 post coitum. Non-mated females were re-paired with alternative partners from the same group following completion of the original pairing period for a maximum of 14 days.

All dams were allowed to give birth and rear their F1 litters up to 21 days post partum. The offspring were examined as soon as possible after completion of the delivery and throughout the lactation period for litter size, live birth, still birth and gross abnormalities. Pups were individually weighed on days 0 (if possible) and/or day 1, 4, 7, 14 and 21 of lactation. The pups were caged together with the dam until weaning on day 21. All dams and pups were observed daily for survival, behavioural abnormalities in nesting and nursing.

On day 4 post partum, the size of each litter was adjusted randomly to (as near as possible) 4 males and 4 females per litter. At weaning on day 21 post partum, all remaining pups and dams were sacrificed. Parental generation males were sacrificed when they were no longer necessary for the assessment of reproductive effects (earliest after day 4 post partum of the F1 pups).

Animals were allocated to their test groups using a computer-generated random algorithm. In addition, body weights were taken into consideration in order to ensure similar mean body weights in all groups.

Results and discussion

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

ca. 300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Increase in post implantation loss and number of dead pups at top dose

2

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No fertility effects seen

3

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No observable maternal toxicity

4

Dose descriptor:

NOAEL

Effect level:

ca. 300 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Paternal toxicity seen at top dose

Observed effects

There were no mortalities or clinical signs observed in parental animals from any group. Males treated with 1000 mg/kg bw/day had significantly decreased food consumption during the first 15 days of the pre-pairing period. Mean body weight and mean body weight gain were also reduced during the first 20 days of the pre-pairing period. Food consumption, body weight and body weight gain of females was not affected by treatment with the test item.
 
For males at 1000 mg/kg bw/day, the mean liver body weight ratio was statistically significantly increased and was considered to be test item related. An increase in post-implantation loss was seen in females treated with 1000 mg/kg bw/day and this was considered to be possibly test item related.
 
There was no increase in mortality or clinical signs observed in the F1 pups following treatment with the test item. Mean pup weight was not affected by treatment. Additionally, during the macroscopic examination of pups no abnormal findings that were considered to be related to treatment with the test item were noted. In females, the post implantation loss and number of dead pups were increased at 1000 mg/kg bw/day.

Any other information on results incl. tables

Table 2: Summary of food consumption of males

Days	Group 1 (0 mg/kg bw/day)		Group 2 (100 mg/kg bw/day)		Group 3 (300 mg/kg bw/day)		Group 4 (1000 mg/kg bw/day)
	Food consumption (g)	% of control	Food consumption (g)	% of control	Food consumption (g)	% of control	Food consumption (g)	% of control
Pre-pairing period
1 - 8	23.6 ± 1.5	-	23.4± 1.3	-0.8	22.9 ± 1.3	-3.0	22.5 ± 1.1**	-4.7
8 - 15	24.4 ± 1.7	-	24.1 ± 1.6	-1.2	23.6 ± 1.9	-0.8	22.8 ± 1.4**	-0.8
15 - 22	24.4 ± 1.9	-	24.2 ± 2.0	-0.8	23.7 ± 1.8	-2.9	23.7 ± 1.4	-2.9
22 - 29	24.2 ± 2.0	-	24.0 ± 1.9	-0.8	23.7 ± 1.8	-2.1	23.7 ± 1.5	-2.1
29 - 36	23.9 ± 2.1	-	24.2 ± 1.7	-1.3	23.7 ± 1.9	-0.8	23.7 ± 1.3	-0.8
36 - 43	23.8 ± 1.6	-	24.0 ± 1.7	-0.8	23.6 ± 2.0	-0.8	23.7 ± 1.3	-0.4
43 - 50	23.6 ± 1.9	-	23.4 ± 1.7	-0.8	23.3 ± 1.7	-1.3	23.6 ± 1.3	+0.0
50 - 57	24.5 ± 2.0	-	24.6 ± 2.0	-0.4	24.4 ± 2.1	-0.4	25.1 ± 1.4	+2.4
57 - 64	24.2 ± 1.9	-	24.3 ± 1.7	+0.4	24.2 ± 1.9	+0.0	24.3 ± 1.3	+0.4
64 - 70	24.7 ± 2.0	-	24.6 ± 1.8	-0.4	24.5 ± 1.9	-0.8	24.8 ± 1.5	+0.4
After pairing period
1 - 8	23.9 ± 3.3	-	24.3 ± 1.7	+1.7	24.6 ± 2.0	+2.9	24.4 ± 1.4	+2.1
8 - 15	24.2 ± 2.0	-	23.5 ± 1.7	-2.9	24.2 ± 1.9	+0.0	23.7 ± 1.4	-2.1
15 - 19	24.1 ± 2.4	-	24.0 ± 1.8	-0.4	24.8 ± 2.5	+2.9	24.9 ± 1.5	+3.3

* p<0.05, ** p<0.01

 

 

Table 3: Summary of food consumption of females 

Days	Group 1 (0 mg/kg bw/day)		Group 2 (100 mg/kg bw/day)		Group 3 (300 mg/kg bw/day)		Group 4 (1000 mg/kg bw/day)
	Food consumption (g)	% of control	Food consumption (g)	% of control	Food consumption (g)	% of control	Food consumption (g)	% of control
Pre-pairing period
1 - 8	18.0± 1.2	-	17.5± 1.4	-2.8	17.2± 1.2	-4.4	17.2± 1.2	-4.4
8 - 14	18.7± 1.4	-	18.3± 1.7	-2.1	17.8± 1.3	-4.8	18.1± 1.4	-4.4
Gestation period
1 - 7	22.2± 2.7	-	22.2± 2.0	+0.0	22.4± 2.1	+0.9	22.5± 1.9	+1.4
7 - 14	24.1± 2.6	-	24.0± 2.1	-0.5	24.1± 2.1	+0.0	24.5± 2.2	+1.7
14 - 21	25.7±2.4	-	25.0± 2.6	-2.7	25.9± 1.9	+0.8	26.0± 2.1	+1.1
Lactation period
1 - 7	36.0± 3.9	-	36.5± 6.0	+1.4	36.4± 3.8	+1.1	36.6± 4.6	+1.7
7 - 14	55.6± 4.3	-	54.7± 8.7	-1.6	57.2± 3.9	+2.9	54.4± 5.0	-2.2

 

 

Table 4: Summary of body weights (g) and body weight gain (%) of males 

Days	Group 1 (0 mg/kg bw/day)		Group 2 (100 mg/kg bw/day)		Group 3 (300 mg/kg bw/day)		Group 4 (1000 mg/kg bw/day)
	Body weight (g)	Body weight gain (%)	Body weight (g)	Body weight gain (%)	Body weight (g)	Body weight gain (%)	Body weight (g)	Body weight gain (%)
Pre-pairing period
1	203 ± 9.4	0	203± 8.1	0	201 ± 8.5	0	202± 8.1	0
8	243 ± 11.9	19	241 ± 10.2	19	237 ± 14.9	18	236 ± 11.2	17*
15	276 ± 15.3	36	272 ± 14.2	34	267 ± 21.2	33	262 ± 16.1*	30**
22	303 ± 18.9	49	300 ± 19.0	48	296 ± 25.8	47	289 ± 18.8	43
29	325 ± 21.1	60	322 ± 22.0	59	319 ± 28.6	59	311 ± 21.5	54
36	342 ± 23.3	68	339 ± 23.8	67	326 ± 32.0	67	327 ± 23.1	62
43	357 ± 25.0	76	354 ± 26.0	75	353 ± 36.2	76	345 ± 23.8	71
50	369 ± 25.9	82	366 ± 28.1	81	365 ± 36.8	82	356 ± 25.5	76
57	383 ± 27.9	88	380 ± 29.9	88	381 ± 38.0	89	369 ± 25.5	83
64	393 ± 29.8	94	389 ± 30.4	92	391 ± 39.6	95	378 ± 27.9	87
70	401 ± 30.6	97	396 ± 31.4	96	399 ± 40.8	99	384 ± 28.7	90
Pairing period
1	398 ± 31.4	0	392 ± 31.1	0	395 ± 42.7	0	379 ± 28.8	0
8	406 ± 30.5	2	401 ± 31.2	2	407 ± 40.4	3*	390 ± 28.0	3*
15	414 ± 31.9	4	409 ± 31.7	4	418 ± 41.5	6**	399 ± 31.0	5
18	418 ± 31.7	5	414 ± 32.2	6	422 ± 41.7	7**	403 ± 30.7	6
After pairing period
1	419 ± 32.1	0	415 ± 32.3	0	423 ± 41.8	0	403 ± 29.9	0
8	422 ± 33.1	1	419 ± 33.2	1	428 ± 42.9	1	402 ± 30.1	0
15	428 ± 33.6	2	427 ± 34.2	3	436 ± 43.5	3	411 ± 30.1	2
19	432 ± 36.1	3	430 ± 35.3	4	440 ± 44.8	4	412 ± 29.5	2

* p<0.05, ** p<0.01

 

Table 5: Summary of body weights (g) and body weight gain (%) of females 

Days	Group 1 (0 mg/kg bw/day)		Group 2 (100 mg/kg bw/day)		Group 3 (300 mg/kg bw/day)		Group 4 (1000 mg/kg bw/day)
	Body weight (g)	Body weight gain (%)	Body weight (g)	Body weight gain (%)	Body weight (g)	Body weight gain (%)	Body weight (g)	Body weight gain (%)
Pre-pairing period
1	157 ± 9.4	0	159± 6.1	0	157 ± 6.1	0	157 ± 8.7	0
8	176 ± 10.1	12	175 ± 8.1	11	174 ± 8.3	11	173 ± 9.4	10
14	188 ± 12.7	20	186 ± 7.9	18	184 ± 9.1	17	186 ± 9.8	18
Pairing period
1	193 ± 13.4	0	193 ± 8.1	0	192 ± 8.1	0	191 ± 8.1	0
8	208	17	205 ± 9.4	10	-	-	-	-
15	-	-	218	13	-	-	-	-
18	-	-	229	19	-	-	-	-
Gestation period
1	195 ± 12.2	0	197 ± 10.4	0	192 ± 10.9	0	193 ± 8.9	0
8	230 ± 15.1	18	231 ± 13.5	17	229 ± 13.5	20	229 ± 11.8	18
15	264 ± 20.1	36	264 ± 15.9	34	262 ± 15.8	37	264 ± 15.9	37
19	308 ± 23.5	58	307 ± 21.3	56	309 ± 18.2	62	309 ± 20.3	60
Lactation period
1	240 ± 19.7	0	237 ± 14.3	0	241 ± 15.9	0	244 ± 13.8	0
8	268 ± 19.2	12	269 ± 19.0	13	269 ± 13.4	12	270 ± 13.7	11
15	288 ± 19.8	20	291 ± 19.1	23	290 ± 14.0	21	291 ± 17.8	19
21	280 ± 19.1	17	286 ± 16.8	21*	288 ± 12.0	20	291 ± 15.8*	19

Table 6: Breeding data per group 

	Group 1 (0 mg/kg bw/day)	Group 2 (100 mg/kg bw/day)	Group 3 (300 mg/kg bw/day)	Group 4 (1000 mg/kg bw/day)
Litters	23	24	23	24
Duration of gestation (days)	21.5 ± 0.51	21.5 ± 0.51	21.3 ± 0.49	21.5 ± 0.51
Implantations	13.0 ± 2.04	12.9 ± 2.97	13.2 ± 1.91	13.3 ± 1.78
Post implantation loss	0.5 ± 0.95	0.7 ± 0.86	0.7 ± 0.88	1.8 ± 1.86*
Dead pups at first litter check: Litters affected (#) Total number of pups	1 1	0 0	1 1	2 8
Living pups at first litter check % of males/females Mean ± SD	53/47 12.5 ± 2.02	47/53 12.2 ± 2.79	45/55* 12.6 ± 1.80	45/55* 11.5 ± 2.55
Postnatal loss days 0-4 p.p. as % of living pups Litters affected (#) Total number of pups	1.0 3 3	0.3 1 1	2.4 5 7	1.5 3 4
Breeding loss days 5-21 p.p. as % of living pups Litters affected (#) Total number of pups	0.0 0 0	0.5 1 1	0.0 0 0	0.0 0 0
Birth index	96.0	94.5	95.1	86.5**
Viability index	99.0	99.7	97.6	98.5
Weaning index	100.0	99.5	100.0	100.0

 

Applicant's summary and conclusion

Conclusions:

The critical NOAEL for parental animals and offspring was established at 300 mg/kg bw/day, based on reductions in food consumption, body weight gain and liver body weight ratio in paternal males, and increased post-implantation loss and number of dead pups in maternal females at the top dose (1000 mg/kg bw/day). No maternal toxicity, fertility or teratogenic effects were seen at any dose with RA2

Executive summary:

The potential of RA2 to induce reproductive toxicity was investigated in a one-generation reproduction toxicity study. RA2 was administered to groups of Wistar rats (24/sex/dose) at the following doses: 0, 100, 300 and 1000 mg/kg bw/day. The test item was administered once daily to males for a 70 day pre-pairing period, during the pairing period and until the last litter had reached day 4 post partum. Females received the test item during a 14 day pre-pairing period and also during the pairing, gestation and lactation periods.

 

Parental rats were regularly monitored for mortality and clinical signs twice a day. Body weight was recorded daily from the start of treatment until necropsy. Food consumption was recorded weekly. All parental animals were subjected to gross necropsy, with particular attention to the organs of the reproductive system. Histopathological observations were carried out on the following organs for all high dose and control parental animals: pituitary glands, adrenals, uterus, oviducts, cervix, ovaries, testes with epididymides, prostate, seminal vesicles with coagulating glands, vagina, target organs. Histopathology was also performed on the reproductive organs of all infertile animals and on all gross lesions from all animals.

 

Reproductive and litter parameters assessed included: pregnant females during the first/second pairing period, number of females delivering pups, not mated/not pregnant, number of females rearing pups to weaning, duration of gestation, implantation rate, post-implantation loss, litter size, postnatal loss, breeding loss, abnormal findings at birth/during lactation/at macroscopic examination, pup weights after birth and sex ratios.

 

There were no mortalities or clinical signs observed in parental animals from any group. Males treated with 1000 mg/kg bw/day had significantly decreased food consumption during the first 15 days of the pre-pairing period. Mean body weight and mean body weight gain were also reduced during the first 20 days of the pre-pairing period. Food consumption, body weight and body weight gain of females was not affected by treatment with the test item.  For males at 1000 mg/kg bw/day, the mean liver body weight ratio was statistically significantly increased and was considered to be test item related.

No effects on fertility were seen at any dose and no treatment-related microscopic changes were seen in parental animals (including the reproductive organs) or their offspring, compared to controls.

At 1000 mg/kg bw/day, an increase in post-implantation loss and increase in the number of dead pups was seen; and this was considered to be "possibly test-item related".

There was no increase in mortality or clinical signs observed in the F1 pups following treatment with the test item. Mean pup weight was not affected by treatment. Additionally, during the macroscopic examination of pups no abnormal findings that were considered to be related to treatment with the test item were noted.

Overall, the critical NOAEL for parental animals and F1 pups was established at 300 mg/kg bw/day, based on reductions in food consumption, body weight gain and liver body weight ratio in males, and increased post-implantation loss and number of dead pups in females at the top dose (1000 mg/kg bw/day). No maternal toxicity, fertility or teratogenic effects were seen at any dose.