Registration Dossier

There are in vitro studies trying to show developmental effects. We can note that in Spezia (1992) human S9 did not behave as rat S9 to induce effects which are not similar to in vivo for ASA.

Reference

Endpoint:

three-generation reproductive 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:

supporting study

Study period:

no data are available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study is similar to OECD 416, several currently recommended parameters were not assessed, but the study 2 years/oral/rat (Webb and Hansen, 1963 (reliability: 2) was used to supplement some observations.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

several deficiencies in relation to OECD Guideline 416 in terms of parameters studied

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Osborne-Mendel

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: (P) x wks; (F1) x wks
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: no data
- Diet: ad libitum (Purina Laboratory Chow)
- Water: ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

IN-LIFE DATES: no data

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): the diet was prepared every 14 days in a manner identical to that of Webb and Hansen (1963) and according to the results of Jones et al (1962).
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: no data

VEHICLE: none

Details on mating procedure:

no data are available

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

none

Duration of treatment / exposure:

100 days before the first mating and then throughout the experiment.

Frequency of treatment:

once/day

Details on study schedule:

no data are available.

Remarks:

Doses / Concentrations:
0, 500, 1500, 3000 and 5000 ppm (equivalent to 25, 75, 150, 250 mg/kg bw as MeS, or 22.5, 67.5, 135, 225 mg/kg bw as SA)
Basis:
nominal in diet

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent no treatment

Details on study design:

no data are available

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: No

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): no

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter ; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
number of pups, stillbirths, live births, presence of gross anomalies,

GROSS EXAMINATION OF DEAD PUPS: no

Postmortem examinations (parental animals):

not performed

Postmortem examinations (offspring):

SACRIFICE: no data

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations for the third generation only.

HISTOPATHOLOGY :
microscopic examination of livers and kidneys was performed.

Statistics:

the Chi-2 test was used to determine significant differences between each dose and the control for each mating in each generation.

Reproductive indices:

the fertility index (number of litters cast/number of females exposed to mating).

Offspring viability indices:

the viability index (number of liveborn/total number born)
the survival index (number alive at day 4/ number born alive)
the weaning index (adjusted number of day 21 survivors/number alive at day 4)

Clinical signs:

no effects observed

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS:
No clinical signs of toxicity were reported.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):

- Fertility index: no significant differences for any dose/1st generation. Appreciable decreases seen in 2nd and 3rd generations/5000 ppm.
- Average litter size/female: significant decreases were seen in the second generation in the second mating at 3000 ppm and in both mating at 5000 ppm. Although decreases were seen at 1500 ppm, they were not statistically significant because of the large variation in progeny between females within a group.

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Sex:

male/female

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Sex:

male/female

Remarks on result:

other: Generation: reproduction (migrated information)

Dose descriptor:

LOAEL

Effect level:

150 mg/kg bw/day

Remarks on result:

other: Generation: development (migrated information)

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day

Remarks on result:

other: Generation: development (migrated information)

Clinical signs:

not examined

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING)

- The average number of liveborn young per female exposed to mating: Statistically significant differences were observed in both matings of the
second generation at 3000 ppm and at 5000 ppm.

- Viability index: "possible loss of young through stillbirths" in 2 matings/5000 ppm.

- Average no.surviving progeny/female, day 4: significant decreases occurred in both matings of the second generation at 3000 ppm and 5000 ppm

- Survival index, day 4: an adverse effect was observed in the second generation at the 3000 and 5000 ppm and in the first mating of the third generation at the same dose levels.

- Average no. progeny weaned/female, day 21: significant decreases were observed in the second generation at 3000 ppm in the first mating and at 5000 ppm in the first and second matings.

- Weaning index: "appreciable decrease" in 2nd generation/2nd litter/5000 ppm.

BODY WEIGHT:

- Average weanling weight,( day 21/sex): decreases in weight appeared consistently at the 3000 and 5000 ppm levels(in all generations).
GROSS PATHOLOGY (OFFSPRING)
no grossly visible abnormalities.

HISTOPATHOLOGY (OFFSPRING)
Histopathological examinations of the livers and kidneys of 3rd weanlings at 0, 3000 and 5000 ppm dose levels showed no indication of toxic effects

Reproductive effects observed:

not specified

Supplemental study: the results obtained after addition of calcium carbonate to methyl salicylate did not differ from those obtained after administration of methyl salicylate alone.

table 1 : fertility indexes of rats fed methyl salicylate for 3 generations

dietary level (ppm)
		0		500		1500		3000		5000
generation	mating	FI (a)	% (b)	FI	%	FI	%	FI	%	FI	%
1	1	20/20	100	20/20	100	20/20	100	20/20	100	20/20	100
	2	19/19	100	20/20	100	18/19	95	19/19	100	20/20	100
2	1	20/20	100	19/20	95	20/20	100	19/20	95	17/20	85
	2	19/19	100	19/20	95	19/19	100	19/20	95	10/13	77
3	1	20/20	100	18/20	90	18/19	95	19/20	95	17/19	89
	2	18/20	90	16/18	89	17/19	89	15/17	88	16/19	84

(a) Fertility Index (nb of litters cast/ nb of females exposed to mating)

(b) Percent females pregnant

table 2: average litter size of rats fed methyl salicylate for 3 generations

dietary level (ppm)
		0		500		1500		3000		5000
generation	mating	No. (a)	Av. (b)	No.	Av.	No.	Av.	No.	Av.	No.	Av.
1	1	208/20	10.4	211/19	11.1	207/20	10.4	235/20	11.8	188/18	10.4
	2	213/19	11.2	232/20	11.6	228/19	12.0	238/19	12.5	198/19	10.4
2	1	216/20	10.8	205/20	10.2	206/20	10.3	169/20	8.4	124/20	6.2 (c)
	2	226/19	11.9	204/20	10.2	189/18	10.5	187/20	9.4 (d)	86/13	6.6 (c)
3	1	192/20	9.6	188/19	9.9	172/19	9.1	170/20	8.5	179/19	9.4
	2	197/20	9.8	191/18	10.6	163/19	8.6	132/17	7.38	172/19	9.1

(a) Total number progeny/number females exposed to mating

(b) Average litter size per female exposed to mating

(c) significant at P<0.01

(d) significant at P<0.05

table 3: viability data for rats fed methyl salicylate for 3 generations

dietary level (ppm)
		0			500			1500			3000			5000
gen.	mating	No. (a)	Av. (b)	VI (c, d)	No.	Av.	VI (c, d)	No.	Av.	VI (c, d)	No.	Av.	VI (c, d)	No.	Av.	VI (c, d)
1	1	208/20	10.4	1,00	211/19	11.1	1,00	195/20	9,8	0,94	229/20	11,4	0,97	167/18	9,3	0,88
1	2	213/19	11.2	1,00	231/20	11.6	1,00	226/19	11,9	0,99	237/19	12,5	1,00	189/19	9,9	0,95
2	1	215/20	10.8	1,00	203/20	10.2	0,99	203/20	10,2	0,99	164/20	8,2 (e)	0,97	106/19	5,6 (f)	0,85
2	2	225/19	11.8	1,00	203/20	10.2	1,00	189/18	10,5	1,00	182/20	9,1 (e)	0,97	82/13	6,3 (f)	0,95
3	1	188/20	9,4	0,98	184/19	9,7	0,98	160/19	8,4	0,93	164/20	8,2	0,96	174/19	9,2	0,97
3	2	196/20	9.8	1,00	186/18	10,3	0,97	155/19	8,2	0,95	118/17	6,9	0,89	166/19	8,7	0,97

(a) Total number liveborn/number females exposed to mating

(b) Average number liveborn per female exposed to mating

(c) Viability index (no. liveborn/total no. born)

(d) Not analyzed for statistical significance

(e) significant at P<0.05

(f) significant at P<0.01

table 4: survival data of rats fed methyl salicylate for 3 generations

dietary level (ppm)
		0			500			1500			3000			5000
gen.	mating	No. (a)	Av. (b)	SI (c, d)	No.	Av.	SI	No.	Av.	SI	No.	Av.	SI	No.	Av.	SI
1	1	157/17	9,2	0,90	116/14	8,3	0,82	172/19	9,1	0,96	152/15	10,1	0,92	129/15	8,6	0,94
1	2	202/19	10,6	0,95	196/20	9,8	0,85	205/19	10,8	0,91	218/19	11,5	0,92	168/19	8,8	0,89
2	1	188/20	9,4	0,87	179/20	9	0,88	190/20	9,5	0,94	123/20	6,2 (e)	0,75	82/19	4,3 (f)	0,77
2	2	211/19	11,1	0,94	188/20	9,4	0,93	186/18	10,3	0,98	165/20	8,2 (e)	0,91	61/13	4,7 (f)	0,74
3	1	174/20	8,7	0,93	177/19	9,3	0,96	147/19	7,7	0,92	139/20	7	0,85	147/19	7,7	0,84
3	2	174/20	8,7	0,89	179/18	9,9	0,96	150/19	7,9	0,97	113/17	6,6	0,96	153/19	8,1	0,92

(a) Total number day 4 survivors / no. females exposed to mating

(b) Average number day 4 survivors per female exposed to mating

(c) Survival index (no. day 4 survivors/total no. liveborn)

(d) Not analyzed for statistical significance

(e) significant at P<0.05

(f) significant at P<0.01

table 5: weaning data of rats fed methyl salicylate for 3 generations

dietary level (ppm)
		0			500			1500			3000			5000
gen.	mating	No. (a)	Av. (b)	WI (c, d)	No.	Av.	WI	No.	Av.	WI	No.	Av.	WI	No.	Av.	WI
1	1	154/17	9,1	0,98	114/14	8,1	0,98	172/19	9,1	1,00	151/15	10,1	0,99	129/15	8,6	1,00
1	2	183/19	9,6	0,91	187/20	9,4	0,95	203/19	10,7	0,99	191/19	10,1	0,88	164/19	8,6	0,98
2	1	176/20	8,8	0,94	168/20	8,4	0,94	188/20	9,4	0,99	121/20	6 (e)	0,98	74/19	3,9 (f)	0,90
2	2	200/19	10,5	0,95	173/20	8,6	0,92	179/18	9,9	0,96	160/20	8,0	0,97	48/13	3,7 (f)	0,79
3	1	170/20	8,5	0,98	172/19	9,1	0,97	146/19	7,7	0,99	122/20	6,1	0,88	137/19	7,2	0,93
3	2	170/20	8,5	0,97	179/18	9,9	1,00	149/19	7,8	0,99	111/17	6,5	0,98	144/19	7,6	0,94

(a) Total of no. of adjusted day 21 survivors/ no. females exposed. adjusted day 21 survivors = (no. alive at day 21)/ (no. kept at day 4) x no. alive at day 4

(b) Average number adjusted day 21 survivors per female exposed to mating

(c) Weaning index (no. adjusted day 21 survivors/ total no. alive at day 4)

(d) Not analyzed for statistical significance

(e) significant at P<0.05

(f) significant at P<0.01

Conclusions:

Under the test conditions, MeS did not significantly reduce male or female fertility. MeS induced developmental toxicity, adverse effects on offspring viability was observed but with no evidence of increased incidence of malformations at the doses tested.The NOAELs were roughly identified:
NOAEL (parental): 250 mg/kg bw/day
NOAEL (reproduction): 250 mg/kg bw/day
LOAEL (development): 150 mg/kg bw/day
NOAEL (development): 75 mg/kg bw/day

Executive summary:

In a 3 -generation study (Collins et al., 1971), rats were fed methyl salicylate at doses of 500, 1500, 3000 or 5000 ppm in the diet (equivalent to 25, 75, 150 or 250 mg/kg body weight as MeS) 100 days before the first mating and then throughout the experiment. No clinical signs of toxicity were reported at any dose level. No statistically significant decrease was reported in fertility index at any dose, however it was considered that there were "appreciable" decreases at 250 mg/kg in F2 and F3. Significant decreases were reported in average litter size, average number of live-born progeny, average numbers of survivors to PND4 and average number of weaning in the 150 and 250 mg/kg bw/day in F2. No external malformations were reported in pups of any litter and necropsy of the third generation weanlings showed no significant findings. The effects in the calcium carbonate supplement groups did not differ significantly from those of the groups fed MeS alone.

NOAEL (parental): 250 mg/kg bw/day, over the know NOAEL from Webb (1963) of 50 mg/kg.

NOAEL (reproduction): 250 mg/kg bw/day

LOAEL (development): 150 mg/kg bw/day

NOAEL (development): 75 mg/kg bw/day

This study is similar to OECD guideline 416. Several currently recommended observations and parameters determinations were not performed, adult body weight and food consumption were not measured in this study, but were stated to have been unaffected at 5000 ppm in a previous study (Webb and Hansen, 1963), there are no data concerning possible effects in sex organs, corpora lutea, pre-implantation or post-implantation losses for any mating. However, no abnormalities in testes/prostate or ovaries/uterus were found in a 2 years study in rats (Webb and Hansen, 1963). Oestrous cycle data and sperm morphology/function data were not measured. Notwithstanding these deficiencies, the study is acceptable (reliability: 2) for reproductive risk assessment.

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

225 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

See arguments, rat used even if effects are not relevant to humans, but adult NOEAL of 50 mg /kg /d from 28 days to 2 years Me Sal.

Endpoint conclusion:

no study available

Endpoint conclusion:

no study available

Fertility Assessment of the potential of ASA to impair fertility has been based on read-across from MeS. The key study is a 3-generation study in rats (Collins et al, 1971), with supporting 2-generation studies in rats (Abbott, 1978) and mice (NTP 1984). No statistically significant effect on fertility was reported in any study. Reduced embryo-foetal viability was reported at high maternally toxic dose levels. Developmental Toxicity The effect of ASA on development has been studied in rats, mice and rabbits with results leading to the conclusion that there are considerable species differences in sensitivity, with the rat being the most sensitive species. Data on the effect of aspirin (ASA) in human pregnancy has been used to assess the relevance of the animal data for risk assessment. These data indicate that humans are less sensitive than rats to the effect of ASA and more comparable to rabbits in several points including ADME or protein binding. A rat key study (Gupta et al, 2003) administered aspirin (ASA) on GD6-17. Results from all studies showed that acetyl salicylic acid is embryotoxic at moderate maternal toxic doses and induces malformations at high maternally toxic doses. For effects in rabbits, the key study is Cappon et al (2003). There were no adverse effects on development at doses not causing severe maternal toxicity. Well-designed epidemiological studies (Slone, 1976;Shapiro, 1976; Kozer, 2002)on the use of aspirin at up to the maximum recommended therapeutic dose of 4000 mg/day (equivalent to 66.7 mg/kg bw/day as ASA or 51 mg/kg/day as SA) have largely demonstrated an absence of increased risk of adverse pregnancy outcome in terms of frequency of stillbirth, neonatal mortality, birth defects or developmental delay, despite widespread self-administration of aspirin during pregnancy. A meta-analysis of studies on the use of low-dose aspirin at 50-150 mg/day (Kozer, 2003) has demonstrated that this dose range is not associated with any adverse pregnancy outcomes, in terms of perinatal mortality, birth complications, congenital malformations or adverse effect on subsequent development. For pregnancies where there was moderate or high risk of pre-eclampsia and/or premature delivery, adverse pregnancy outcome rate was reduced with low-dose aspirin. There was no increased risk of early miscarriage with this dose regime. These data have been reviewed and completed by an Epidemiologist (Pr. D.BARD report to Novacyl, 2012) with a conclusion of no link between ASA use during pregnancy and reprotoxic effects. This absence of any clear evidence of adverse effects from aspirin on human development demonstrated in well-designed epidemiological studies despite widespread prescribed use and self-medication with aspirin at all stages of pregnancy over a period spanning several decades appears to indicate that humans are considerably less sensitive(ordifferent) than rats to the developmental toxicity of salicylate. Overall, it can be concluded that ASA does not adversely affect fertility and that the developmental toxicity reported in the rat is of very questionable (significance/ relevance) for humans. Since, ASA is not considered as reprotoxic for humans, no DNEL should be derived for this endpoint, the rabbit NOAEL being higher than the rat used..

By using the MeS data on rats and deriving the long term DNEL, it covers any potential reproductive effects.

Short description of key information:
No fertility studies are available on acetylsalicylic acid (ASA) itself. Assessment of the potential of ASA to impair fertility has been based on read-across data from published data on related salicylates.
The key study for this endpoint is a 3-generation reproductive toxicity study in Osborne-Mendel rats on Methyl salicylate (Collins et al, 1971).
No statistically significant decrease was reported in fertility index at any dose for any generation. Adverse effects were reported on offspring,
representing embryo-foetal toxicity primarily in terms of reduced viability.

Justification for selection of Effect on fertility via oral route:
3 generations study

Justification for selection of Effect on fertility via inhalation route:
Not a route of exposure due to granulometry of ASA

Justification for selection of Effect on fertility via dermal route:
Not a route of exposure due very low dermal penetration of ASA.

.A key study (Gupta et al , 2003) administered aspirin (ASA) on GD6-17.  Results from all studies showed that salicylic acid is embryotoxic at moderate maternal toxicdoses and induces malformations at high maternally toxic doses For effects in rabbits, the key study is Cappon et al (2003).  There were no adverse effects on development at doses not causing severe maternal toxicity.Well-designed epidemiological studies (Slone, 1976;Shapiro, 1976; Kozer, 2002)on the use of aspirin at up to the maximum recommended therapeutic dose of 4000 mg/day (equivalent to 66.7 mg/kg bw/day as ASA or 51 mg/kg/day as SA) have largely demonstrated an absence of increased risk of adverse pregnancy outcome in terms of frequency of stillbirth, neonatal mortality, birth defects or developmental delay, despite widespread self-administration of aspirin during pregnancy.  A meta-analysis of studies on the use of low-dose aspirin at 50-150 mg/day (Kozer, 2003) has demonstrated that this dose range is not associated with any adverse pregnancy outcomes, there was no increased risk of early miscarriage with this dose regime. These data have been reviewed and completed by an Epidemiologist (Pr. D.BARD report to Novacyl, 2012) with a conclusion of no link between ASA use during pregnancy and reprotoxic effects.
This absence of any clear evidence of adverse effects from aspirin on human development demonstrated in well-designed epidemiological studies despite widespread prescribed use and self-medication with aspirin at all stages of pregnancy over a period spanning several decades appears to indicate that humans are considerably less sensitive than rats to the developmental toxicity of salicylate. 
Overall, it can be concluded ththat ASA does not adversely affect fertility and that the developmental toxicity reported in the rat is of very questionable significance/ relevance for humans. 
Since, ASA is not considered as reprotoxic for humans, no DNEL should be derived for this endpoint.

Reference

Endpoint:: developmental toxicity
Type of information:: experimental study
Adequacy of study:: key study
Study period:: no data are available
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: other: No GLP stated, purity unknown but this recent study is performed in an ICH guideline (CPMP/ICH/386/95) which requires compliance with GLP or equivalent standard.
Reason / purpose for cross-reference:: reference to other study
Qualifier:: according to guideline
Guideline:: other: ICH Topic S 5(R2)
Principles of method if other than guideline:: ASA was administered to pregnant New Zealand rabbits from gestation days GDs 7 to 19 (comparable to guideline 414) and as single high doses on GD 9, 10 or 11. Cesarean sections were performed on GD29, and the fetuses were examined for external, visceral, and skeletal development.
GLP compliance:: not specified
Limit test:: no
Species:: rabbit
Strain:: New Zealand White
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Covance Research Products, Inc (Denver, CO)
- Age at study initiation: 5 to 6.5 months
- Weight at study initiation: 2.8 kg
- Fasting period before study: 1 day
- Housing: rabbits were housed in stainless steel suspended wire cages.
- Diet: Purina Regular Rabbit Chow (Ralston, VA)
- Water: ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C ): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs dark /12 hrs light

IN-LIFE DATES: no data
Route of administration:: oral: gavage
Vehicle:: other: methylcellulose
Details on exposure:: PREPARATION OF DOSING SOLUTIONS:
no data

DIET PREPARATION
no data

VEHICLE
- Justification for use and choice of vehicle: no data
- Amount of vehicle (if gavage): 0.5%
- Purity: no data
- Source: Dow Chemical Co., Midland, MI
Analytical verification of doses or concentrations:: no
Details on analytical verification of doses or concentrations:: none
Details on mating procedure:: no data are available
Duration of treatment / exposure:: For the multiple study: from GD7 to GD19
For the single study: individual days, DG9, 10 or 11
Frequency of treatment:: Single daily doses
Duration of test:: around the time of gestation (29 days)
Remarks:: Doses / Concentrations:
For the multiple study: 125, 250 or 350 mg/kg bw/day (96, 192, 268 mg/kg as SA)
Basis:
nominal conc.
Remarks:: Doses / Concentrations:
For the single study: 500, 750 and 1000 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:: For the multiple study: 20
For the single study: 10
Control animals:: yes, concurrent no treatment
Details on study design:: - Dose selection rationale:

For the multiple study: dose levels were based on findings from a dose range-finding study in pregnant rabbits. MTD = 350 mg/kg per day (3/20 animals died as result of treatment) for this dosing duration (13 days) in pregnant rabbits.

For the single study: ASA was administered to rabbits at higher dose > MTD = 350 mg/kg per day (during sensitive periods of cardiovascular development and midline closure)
Maternal examinations:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: all animals were observed at least twice daily for morbidity and mortality during the study.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: it was determined on the day of arrival and then daily beginning on GD8.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: it was determined on the day of arrival and then daily beginning on GD8.
- 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 29
- Organs examined: the abdominal, thoracic and pelvic viscera , Uterus and ovaries
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
Fetal examinations:: - External examinations: Yes: all per litter
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: No
Statistics:: several types of analyses were used for determining dose-response relations to ASA treatment:
Welch trend test (linear and in proportions) and analysis of covariance.
Indices:: no data are available
Historical control data:: no data are available
Details on maternal toxic effects:: Maternal toxic effects:yes

Details on maternal toxic effects:
In the repeated dose study, three does from the 350 mg/kg group died between GD14 and GD16. An additional doe in the 350 mg/kg group aborted on GD22.Necropsy examinations showed evidence of gastrointestinal toxicity(GI) for these does, consisting of dark red foci or pitted areas on the glandular mucosa of the stomach. Maternal body weight gain was significantly reduced in the mid and high dose groups from GD7 to GD13. Food consumption was significantly reduced throught the exposure duration in these groups.
In the single dose study, one doe from the 500 mg/kg group and one from the group 1000 mg/kg group that were dosed on GD10 and two does from the 750 mg/kg group dosed on GD 11 showed signs of GI toxicity. In this study, maternal toxicity was exhibited at all doses by reductions in body weight gain and food consumption for 3 days after treatment.
Dose descriptor:: NOAEL
Effect level:: 125 mg/kg bw/day
Basis for effect level:: other: maternal toxicity
Dose descriptor:: NOAEL
Effect level:: 250 mg/kg bw/day
Basis for effect level:: other: developmental toxicity
Dose descriptor:: NOAEL
Effect level:: 350 mg/kg bw/day
Basis for effect level:: other: other:
Details on embryotoxic / teratogenic effects:: Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
In the repeated dose study, fetal body weight was significantly reduced at 350 mg/kg per day.
Fetal body weights were not affected by single doses of ASA on GD9, 10 or 11.
There were no treatment related external, visceral, or skeletal malformations associated with ASA administration throughout organogenesis (GD7 to 19) or single dose administered during critical developmental windows (even from treatment at up to 1000 mg/kg ).
Abnormalities:: not specified
Developmental effects observed:: not specified
Conclusions:: Under the test conditions, ASA did not induce malformations in rabbits when it was administered as a single dose or during the period of organogenesis (GD7 to 19), even at doses causing significant maternal toxicity. The NOAELs were identified: NOAEL (maternal): 125 mg/kg bw/day NOAEL (malformations): 350 mg/kg bw/day NOAEL (development): 250 mg/kg bw/day.
Executive summary:: In an ICH-compliant study, Acetylsalicylic acid (ASA) was administered by oral gavage to pregnant New Zealand White rabbits at 125, 250 or 350 mg/kg bw/day (96, 192, 268 mg/kg as Salicylic acid (SA)), followed by termination on GD29 (Cappon and al, 2003). 3 does from the high and one doe from the mid dose died between GD14 and 16 and an additional doe aborted on GD22. Necropsy showed evidence of gastrointestinal toxicity. Maternal body weight gain was significantly reduced in the mid and high dose groups from GD7 to GD13. Food consumption was also reduced in these groups. There were no treatment-related effects on corpora lutea, implantation sites, pre-implantation losses or embryofoetal mortality. Mean foetal weight was significantly reduced in the high dose group. There were no treatment-related visceral or external anomalies. The same study also administered ASA at higher doses on individual days, DG9, 10 or 11.No treatment-related malformations were induced in rabbit fetuses by single dose administrations. The NOAELs were identified: NOAEL (maternal): 125 mg/kg bw/day NOAEL (malformations): 350 mg/kg bw/day NOAEL (development): 250 mg/kg bw/day.

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

350 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Good, ICH study.

Endpoint conclusion:

no study available

Endpoint conclusion:

no study available

The effects of salicylic acid, acetylsalicylic acid or sodium salicylate on organogenesis have been investigated in a large number of studies in several animal species, using a variety of protocols. Many are mechanistic studies, using a single, often high, dose on a restricted number of gestation days. Relatively few are comparable to the prenatal developmental toxicity study OECD guideline 414. For Salicylic acid (SA) itself, two studies in rat (Tanaka et al, 1973a and Tanaka et al 1973b) are acceptable as key studies, although SA was administered only from GD8 to GD14. To complement these studies and to provide key data on developmental toxicity in the rabbit, two recent developmental toxicity studies on read-across substance Acetylsalicylic acid (ASA, aspirin) in rats (Gupta et al, 2003) and rabbits (Cappon et al, 2003) have been included as key studies. These studies complied with current ICH guidelines for pharmaceuticals.

In a pre-natal developmental toxicity study (Tanaka et al., 1973a), salicylic acid was administered to pregnant Wistar rats at levels of 0.06, 0.1, 0.2 and 0.4 % in the diet (30, 50, 100, 200 mg/kg bw/day) on GD 8-14. The high dose of 0.4% caused maternal toxicity, high foetal mortality, growth retardation and a high frequency of complex anomalies including cranioschisis, myeloschisis, pes varus, oligodactyly. At 0.2%, significant foetal growth retardation and a low frequency of anomalies. No effect levels were NOAEL (maternal): 0.2% (100 mg/kg bw/day) and NOAEL (development): 0.1% (50 mg/kg bw/day).A parallel study by gavage (Tanaka, 1973b) at 75, 150 and 300 mg/kg bw gave similar results, with no effect levels NOAEL (maternal): 150 mg/kg and NOAEL (development): 75 mg/kg bw.

In a clinical segment II study, ASA was administered by oral gavage to pregnant Sprague-Dawley rats at 50, 125 or 250 mg/kg bw/day (equivalent to 38, 96, 192 mg/kg bw as SA) during organogenesis (GD6 -17) (Gupta & al, 2003). There was a dose-related reduction in maternal bodyweight gain, significant in the mid and high dose groups. At 250 mg/kg bw/day, ASA induced increases in early resorptions, increased post-implantation loss, increased variations and malformations. At 125 mg/kg, foetal viability was reduced.

A number of valid supporting studies in rats report similar results to those described in the key rat studies above. Fritz and Giese (1990), showed a marked increase in embryonic and foetal mortality, delayed ossification and malformations at 180 mg/kg NaS on GD 6-15. Nakatsuka and Fujii (1979), treated SD rats with ASA on GD 7-17. At 200 mg/kg the number of resorptions and malformed survivors were significantly increased. At 100 and 200 mg/kg the average body weights were significantly reduced in a dose-related manner. Schardein et al. (1969) showed ASA to be embryotoxic to rats fed doses of 250 mg/kg bw/day by gavage, or 0.2 or 0.4% (99 or 240 mg/kg bw/day) in the diet on DG 6-15.. These doses caused significant reduction in maternal bodyweight gain. At 224 or 250 mg/kg ASA, all pups were resorbed. There were a number of skeletal malformations in the pups at 99 mg/kg bw/day.

The results of the key and supporting studies in rats demonstrate that SA has an embryofoetotoxic effect in rats at doses not causing clear maternal toxicity, with evidence of malformations at maternally toxic doses.

Potential for peri- and post-natal developmental toxicity has been reported in section 7.8.1 under the multi-generation studies on the read-across substance Methyl salicylate (MeS) and in section 7.8.3, under segment III studies on aspirin (ASA). As described in these sections, high doses of salicylate increased perinatal mortality in rats, but did not affect growth or development of survivors.

ASA was administered by oral gavage to pregnant New Zealand White rabbits at 125, 250 or 350 mg/kg bw/day on GD7-19 (Cappon & al, 2003). Maternal body weight gain was significantly reduced in the mid and high dose groups from GD7 to GD13. Food consumption was also reduced in these groups. Three high dose does and one mid dose doe died during the study. There were no treatment-related effects on corpora lutea, implantation sites, pre-implantation losses or embryofoetal mortality. There were no treatment-related visceral or external anomalies. Reduction in mean foetal weight at 350 mg/kg bw/day was the only developmental adverse effect reported at this maternally toxic dose.

In a supporting study (Schardein et al, 1969), rabbits received ASA at 200 or 250 mg/kg on DG 6-13 or GD6-18. ASA induced maternal toxicity but no skeletal malformations or other effects on offspring.

It is clear that there are differences in sensitivity between the tested species. From developmental toxicity studies equivalent to OECD guideline 414, the rabbit is seen to be considerably less sensitive than the rat to the developmental toxicity of SA and other salicylates. In the multi-generation studies equivalent to OECD guideline 416, it was also seen that the mouse was less sensitive than the rat in this respect.

The rat data provide a “worst case” for risk assessment, however there is a considerable body of published data on the effects of ASA administered during human pregnancy which indicate that pregnant women and their offspring are relatively insensitive to ASA developmental toxicity.

Justification for selection of Effect on developmental toxicity: via oral route:
Study on rabbit was chosen as being more relevant for humans, due to metabolism as reviewed by Rainsford (2004).

Justification for selection of Effect on developmental toxicity: via inhalation route:
Not a route of exposure due to granulometry

Justification for selection of Effect on developmental toxicity: via dermal route:
Not a route of exposure due to very low dermal penetration of ASA

A number of published studies have been reported on in vitro rat, chicken or mouse embryos exposed to salicylates and their major metabolites, cultured during the critical period of fetal organ development. The results obtained provide evidence that the parent salicylate is largely, if not entirely, responsible for the dysmorphogenic effects observed in rats. Yokoyama et al. (1984) reported facial anomalies such as cleft lip and curly tail in the embryos cultured with salicylic acid. Anomalies induced by salicylic acid were localized malformations. Kosazuma et al. (1995) investigated the effects of salicylic acid on the in vitro development of fetal mouse palates. Salicylic acid inhibited both palatal closure and fusion at 2 mM or lower concentrations, while its metabolites were largely inactive. Karabulut et al. (2000), showed that salicylates decreased all growth and developmental parameters in a concentration responsive manner and postulated that developmental toxicity might be related to interaction with free oxygen radicals.These in vitro studies suggest that the developmental toxicity of tested salicylates is likely to be directly related to the salicylic acid (salicylate) concentration reaching the foetus, rather than to its metabolites.

See also studies on blood concentrations in human and rat and fetuses indicating equivalent concentration for allometric doses. But in vivo the concentration in human fetuses is > adult while it is the contrary in rat showing effects at allometric equivalent doses. Is the rat a relevant species for developmental effects?

Fertility:

Not classified for effects on reproduction (fertility) according to EU and GHS (UN/EU) criteria.

Based on multigeneration studies on the effects of the read-across substance MeS on fertility in rats and mice which indicate that SA does not adversely affect fertility.

Development:

Not classified for effects on reproduction (development) according to EU and GHS (UN/EU) criteria.

Based on a weight of evidence approach assessing data from animal studies and human data on acetylsalicylic acid. Different species show a variation in sensitivity to the developmental toxicity of salicylates, including SA. The rat is the most sensitive species, demonstrating effects which could be interpreted to classify, if not taking into account the differences noted with human and repeated toxicity NOAELs. On the other hand, salicylates do not induce developmental effects in the rabbit even at doses causing severe maternal toxicity, and rabbit is more like humans for several imortant points like protein binding and ionisation. An extensive analysis of data on ASA in human pregnancy indicate that humans are relatively insensitive, allowing the conclusion that SA should not be considered a developmental toxicant in humans.

Classification of ASA is therefore not required.