Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information
The substance homosalate was investigated for androgen receptor binding and effects seen were very weak at high concentration and unspecific. It was concluded that the test substance cannot cause a specific interaction with androgen binding domain of the AR.
In order to investigate the potential affinity of test substance for the estrogen receptor (ER), a classical receptor binding assay using human recombinant ER of α-subtype as receptor source and radiolabeled estradiol as ligand was generated. The physiological estrogen estradiol and the phytoestrogen genistein served as reference compounds with strong and weak affinity for the ER, respectively. Results showed that the test substance has no affinity potential for the ER.
Additionally, groups of immature female rats were administered the test substance once a day at levels of 0 (untreated), 0 (vehicle control), 200 and 1000 mg/kg body weight sub-cutaneously for a period of consecutive tree days. Besides two additional positive control groups containing 0.3 and 1.0 μg/kg 17α-Ethinylestradiol were set and treated same with testing groups. Regarding state of health and general behavior of the animals, there was no difference between the untreated and treated animals of all groups. No treatment related mortality has been found. No toxicologically significant effect on the feed intake was observed. Body weights were not affected by treatment. After necropsy, a dose-dependent enlargement of uteri and an increased uterine wet and blotted weight were observed in the positive controls. However, no effect on uteri was observed at either 200 or 1000 mg/kg test substance treated groups.
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:
weight of evidence
Study period:
no data
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.
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 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 daily
Details on study schedule:
no data 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 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:
Chi-2 test was used to determine significant differences between each dose and the control for each mating in each generation.
Reproductive indices:
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: for methyl salicylate
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: for methyl salicylate
Remarks on result:
other: Generation: reproduction (migrated information)
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day
Basis for effect level:
other: for methyl salicylate
Remarks on result:
other: Generation: development (migrated information)
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Basis for effect level:
other: for methyl salicylate
Remarks on result:
other: Generation: development (migrated information)
Dose descriptor:
NOAEL
Effect level:
430 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: Read-across value for homosalate
Dose descriptor:
NOAEL
Effect level:
430 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: Read-across value for homosalate
Remarks on result:
other: Generation: reproduction (migrated information)
Dose descriptor:
LOAEL
Effect level:
258 mg/kg bw/day
Basis for effect level:
other: Read-across value for homosalate
Remarks on result:
other: Generation: development (migrated information)
Dose descriptor:
NOAEL
Effect level:
129 mg/kg bw/day
Basis for effect level:
other: Read-across value for homosalate
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  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
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:
adverse effect observed
Dose descriptor:
NOAEL
430 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
A 3-generation reproductive toxicity study with the read-across substance methyl salicylate and an OECD 422 screening study with homosalate gave consistent results
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

WoE was done based on the an oral OECD 422 reproduction toxicity screening study with homosalate and a 3-generation oral reproduction toxicity study with the read-across substance methyl salicylate. The two studies gave consistent results with a NOAELs of 430 mg/kg bw/day in the 3-generation study and no evidence of reproductive effects at 120 mg/kg bw/day in the OECD 422 study.

A read-across justification is provided as attachment to IUCLID section 13 respectively as appendix to the CSR.


Short description of key information:
No adverse effects observed in oral OECD 422 screening study with homosalate: 120 mg/kg bw/day
NOAEL parental (for homosalate) from 3-generation oral reproductive toxicity study with read-across substance methyl salicylate: 430 mg/kg bw/day
NOAEL reproduction (for homosalate) from 3-generation oral reproductive toxicity study with read-across substance methyl salicylate: 430 mg/kg bw/day

Justification for selection of Effect on fertility via oral route:
WoE was done based on OECD 422 with homosalate and a 3-generation oral reproductive toxicity study with the read-across substance methyl salicylate

Effects on developmental toxicity

Description of key information
No adverse effects observed in oral OECD 422 screening study with homosalate: 120 mg/kg bw/day
NOAEL parental (for homosalate) from 3-generation oral reproductive toxicity study with read-across substance methyl salicylate: 430 mg/kg bw/day
NOAEL development (for homosalate) from 3-generation oral reproductive toxicity study with read-across substance methyl salicylate: 129 mg/kg bw/day
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
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

WoE was done based on the an oral OECD 422 fertility/development toxicity screening study with homosalate and a 3-generation oral reproduction toxicity study with the read-across substance methyl salicylate. A read-across justification is provided as attachment to IUCLID section 13 respectively as appendix to the CSR.

The two studies gave consistent results with a NOAELs of 129 mg/kg bw/day in the 3-generation study and no evidence of developmental effects at 120 mg/kg bw/day in the OECD 422 study.

Based on the weight of evidence approach assessing data from animal studies and human data on acetylsalicylic acid displayed fully in the disseminated REACH dossier on methyl salicylate, different species show a variation in sensitivity to the developmental toxicity of salicylates, including salicylic acid. The rat is the most sensitive species, demonstrating effects which might lead to classification. On the other hand, salicylates do not induce developmental effects in the rabbit even at doses causing severe maternal toxicity. An extensive analysis of data on acetylsalicylic acid in human pregnancy allows the conclusion that salicylic acid should not be considered a developmental toxicant in humans.


Justification for selection of Effect on developmental toxicity: via oral route:
Based on WoE consideration taking into account results from the OECD 422 screening study with homosalate, results from the 3-generation reproduction toxicity study with the read-across substance methyl salicylate and additional data on developmental toxicity available for acetylsalicylic acid, salicylic acid and other salicylates in several animals species and in humans (see Read-across justification document in IUCLI section 13 respectively in the appendix to the CSR).

Justification for classification or non-classification

Fertility:

Not classified for effects on reproduction (fertility) according to CLP (Regulation EC No 1272/2008) and/or DSD (Directive 67/548/EEC). Based on a weight of evidence approach, a 3 -generation study with the read-across substance methyl salicylate on fertility in rats indicated that methyl salicylate and thus also the metabolite salicylatic acid do not adversely affect fertility.

 

Development:

Not classified for effects on reproduction (development) according to CLP (Regulation EC No 1272/2008) and/or DSD (Directive 67/548/EEC). Based on the weight of evidence approach assessing data from animal studies and human data on acetylsalicylic acid displayed fully in the disseminated REACH dossier on methyl salicylate, different species show a variation in sensitivity to the developmental toxicity of salicylates, including salicylic acid. The rat is the most sensitive species, demonstrating effects which might lead to classification. On the other hand, salicylates do not induce developmental effects in the rabbit even at doses causing severe maternal toxicity. An extensive analysis of data on acetylsalicylic acid in human pregnancy indicates that humans are relatively insensitive, allowing the conclusion that salicylic acid should not be considered a developmental toxicant in humans. Classification is therefore not required.