Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
three-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key 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:
reference to other study
Qualifier:
equivalent or similar to
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 and environmental conditions:
TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- 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 data

DETAILED CLINICAL OBSERVATIONS: No data

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
Basis for effect level:
other: no effect observed
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)
Remarks on result:
other: see below
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

Remarks on result:
other: see below
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  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.
 1 208/20 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

 

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

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

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

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

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

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

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

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

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

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

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

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 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, or 22.5, 67.5, 135, 225 mg/kg bw as SA) 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

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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Results supported by several reliable other studies on the substance itself.
Additional information

The reproductive toxicity (fertility) effects of MeS and other salicylates in several animal species have been reviewed in the published literature. MeS and a number of other salicylate esters have been assessed for their safety as cosmetic ingredients (CIR, 2003), as fragrance ingredients (RIFM 2007; Lapczynski, 2007), as food flavouring agents (EPA, 1997; JECFA, 2001; Adams, 2005) and/or (for MeS) as a pesticide (EPA, 2005). It was concluded that neither MeS nor the other reviewed salicylates represented a safety concern for humans in the applications assessed.

Assessment of the potential of MeS to impair fertility has been based on studies on MeS itself and on Acetylsalicylic acid (ASA). A read-across justification is provided as attachment to IUCLID section 13 respectively as appendix to the CSR.

The potential for MeS to affect male and/or female fertility has been assessed in rats and mice in several multi-generation studies, with the study by Collins et al (1971) considered to be the key study for this endpoint.

In a 3-generation study (Collins et al., 1971), MeS was administered to male and female Osborne-Mendel rats in the diet at 500, 1500, 3000 and 5000 ppm (equivalent to 22.5, 67.5, 135, 225 mg/kg bw as SA). Parental generation rats were fed MeS for 100 days prior to mating, then throughout two mating, gestation and lactation periods. F1 and F1 rats received the test compound throughout the study, which terminated with the weaning of the F3 offspring. 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 (decreases in litter size, number of live-born progeny, number of survivors to PND4 and PND5 and number of survivors to weaning) only at maternally toxic doses, according to the chronic systemic oral NOAEL of 50 mg/kg/day.

In a 2-generation study (Abbott & Harrisson, 1978), male and female Wistar rats received MeS in the diet at 2500 and 5000 ppm (equivalent to 113 and 225 mg/kg bw/day as SA) for 60 days prior to mating, then throughout the study. Each generation of rats was mated twice.  This study reported a non-significant decrease in mating performance for the first generation, and reduced viability of pups.

Abbott and Harrisson also reported data on male and female mice exposed to MeS in the same manner at the same dietary concentration as rats (2500 and 5000 ppm (equivalent to 324 and 648 mg/kg bw/day as SA) from 30 days prior to mating.  No adverse effects were reported on any reproductive parameter.

Two 2-generation studies have been conducted on MeS in CD-1 Mice by gavage according to the NTP continuous breeding protocol (NTP, 1984a, 1984b).  At dose levels of 25, 50 and 100 mg/kg bw/day MeS (22.5, 45 and 90 mg/kg bw/day as SA) for 7 days prior to mating then for a 98 day cohabitation period, no effects were reported on fertility, number of pups per litter, percentages of live pups or pup weight.  Necropsy of F1mice revealed no effects on body or organ weights or sperm motility, density or morphology.  In a second study at the higher dose levels of 100, 250 and 500 mg/kg bw/day (90, 225 and 450 mg/kg bw/day as SA) there was no effect on fertility index.  Reduced pup viability was reported at the high dose, with only a 3% reduction in pup weight at the mid dose level.

As conclusions on above studies, no statistically significant effect on fertility was reported in any study. Reduced embryo-foetal viability was reported at high maternally toxic dose levels, when parental toxicity refers to the systemic NOAELs.

The potential for effects on male and female fertility from ASA was reported by Schardein et al. (1969). This study used only a single dose level of 210 mg/kg ASA (177 mg/kg bw as MeS) by oral gavage as positive control. Male rats were treated for 63 days prior to mating with untreated females. Female rats were treated for 14 days prior to mating with untreated males and up to weaning. ASA did not significantly affect male or female fertility at this dose, which caused moderate bodyweight depression in males and severe bodyweight depression in females.

The studies above show a number of deficiencies in relation to current guidelines in terms of parameters studied, however their results are consistent in concluding that salicylates do not significantly affect fertility. In addition, 2-year chronic toxicity studies (Webb, 1963) in rats and dogs showed no abnormalities in testes/prostate or ovaries/uterus.

The adverse effects on reduced viability of offspring reported primarily in rats represent developmental toxicity rather than reduction of the fertility of either male or female animals. It can therefore be concluded that MeS is not likely to have any significant adverse effect on fertility.

Short description of key information:

The key study for this endpoint is a 3-generation reproductive toxicity study in Osborne-Mendel rats (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. Reduced embryo-foetal viability was reported at high maternally toxic dose levels, when parental toxicity refers to the systemic NOAELs: the NOAEL fertility = 250 mg/kg bw/day is higher than the chronic NOAEL of 50 mg/kg bw/day.

Justification for selection of Effect on fertility via oral route:

Reliability 2, 3-generations study performed on Methyl salicylate itself

Effects on developmental toxicity

Description of key information

ASA (acetylsalicylic acid) 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 (296 mg/kg bw/day as MeS).

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
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:
reference to other study
Qualifier:
according to
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 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 ).
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Basis for effect level:
fetal/pup body weight changes
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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
296 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The reproductive toxicity (development) of several salicylates in several animal species have been reviewed in the published literature.

MeS and a number of other salicylate esters have been assessed for their safety as cosmetic ingredients (CIR, 2003), as fragrance ingredients (RIFM 2007; Lapczynski, 2007), as food flavouring agents (EPA, 1997; JECFA, 2001; Adams, 2005) and/or (for MeS) as pesticide (EPA, 2005). None of the reviews considered that these salicylates represented a safety concern for humans in the applications assessed.

No developmental toxicity studies according to current guidelines are available for MeS itself. Assessment has been made by read-across primarily from studies on SA and ASA. A read-across justification is provided as attachment to IUCLID section 13 respectively as appendix to the CSR.

Based on the weight of evidence approach assessing data from animal studies along with human data on acetylsalicylic acid, different species show a variation in sensitivity to the developmental toxicity of salicylates and salicylic acid.

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 SA, two studies in rat (Tanaka et al, 1973a and Tanaka et al 1973b) are acceptable as reliable rat 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 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.

RAT:

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 were noted. 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/day gave similar results, with no effect levels NOAEL (maternal): 150 mg/kg/day and NOAEL (development): 75 mg SA/kg bw/day, that gives for MeS 83 mg/kg bw/day and ASA 98 mg/kg bw/day. Note that maternal toxicity was only evaluated by final body weight gain.

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 and 55, 138, 275 mg/kg bw as MeS) 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 a dose of 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 (not detailed here) 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 causing clear maternal toxicity in systemic assays, with evidence of malformations only at maternally toxic doses.

Potential for peri- and post-natal developmental toxicity has been reported in IUCLID section 7.8.1 under the multi-generation studies on Methyl salicylate (MeS)and in IUCLID 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.

RABBIT:

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 (268 mg/kg bw/day as SA and 296 mg/kg bw/day as MeS).

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

SPECIES RELEVANCE FOR HUMAN DEVELOPMENTAL TOXICITY ASSESSMENT:

It became clear that there are differences in sensitivity between the tested species. Based on developmental toxicity studies equivalent to OECD guideline 414, the rabbit(Cappon, 2003)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 (NTP, 1984), it was also seen that the mouse was less sensitive than the rat in this regard.

When analysing the ASA data, it was evident from the metabolism (Rainsford, 2004) that the rabbit is more human-like with high protein binding capacity in contrast to the rat with a low one. In fact, in the rabbit (Cappon, 2003) there is no prenatal loss or teratogenic effect at 350 mg/kg/d, a distinctly maternally toxic dose.

In man, like in rabbit, there is a strong binding of SA to plasma proteins. When comparing SA binding in rabbits and rats, it is observed that it is significantly lower in rat. So, as only free SA could be transfered through placenta to embryos, this explains the effects in rats, which were “illustrated” by different blood levels in human and rat adults and embryos. Note also that the visceral yolk sac placenta in rats is an ion-trapping environment for weak acids (another difference between rats and rabbits) resulting in higher embryonal levels of SA in rat foetuses than in rabbit foetuses. (Carney et al., 2004). As further example, as reported in ASA IUCLID file, bone effects were observed in rat, while ASA was used for juvenile arthritis treatment in Human without such effects (Abbott and Harrisson, 1978). This made the weight of evidence that the rat is not a relevant species to extrapolate developmental effects to humans.

Relevance of human data with acetylsalicylic acid has extensively been reviewed (90 references have been analysed, among them some meta-analyses, and discussed) in a report by an epidemiologist expert (Bard, 2012), and concluded to no link with ASA medication.

Even the human subacute high dose of ASA (3000 mg/d or 60mg/kg for 50 kg) which corresponds to an allometric rat dose of 240 mg/kg, is higher than the rat NOAELS and far higher than DNELs. Note than in other regions the subacute human dose could be higher.

Overall, it can be concluded that ASA, and therefore SA and MeS, have no developmental toxicity effects in humanswhen doses applied are representative of actual Human exposure (medical treatment at least, at doses higher than the rat NOAELs).

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 classification or non-classification

Fertility:

Not classified for effects on reproduction (fertility) according to CLP criteria.

Multigeneration studies on the effects of the substance on fertility in rats and mice indicate that it does not adversely affect fertility at any dose at any generation.

 

Development:

Not classified for effects on reproduction (development) according to CLP criteria.

Based on a weight of evidence approach assessing data from animal studies and human data on acetylsalicylic acid concluding that ASA, and therefore MeS, have no developmental toxicity effects in humans when doses applied are representative of actual Human exposure (medical treatment at least, at doses higher than the rat NOAELs).(see Discussion section and Read-across justification in Chapter 13).