Methyl salicylate

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

17.5 mg/m³

Most sensitive endpoint:

repeated dose toxicity

Overall assessment factor (AF):

20

Modified dose descriptor starting point:

NOAEC

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

285 mg/m³

Most sensitive endpoint:

repeated dose toxicity

Overall assessment factor (AF):

5

Modified dose descriptor starting point:

NOAEC

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

6 mg/kg bw/day

Most sensitive endpoint:

repeated dose toxicity

Overall assessment factor (AF):

20

Modified dose descriptor starting point:

NOAEL

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

medium hazard (no threshold derived)

Introduction

In this section, data from all end-points are examined and analyzed in order to determine for which it is relevant and possible to establish a DNEL value. The method followed is that proposed in the guidance for the implementation of REACH (Chapter R.8: Characterisation of dose (concentration)-response for human health, May 2008).

Self Classification:

- Methyl salicylate is self-classified:

- Category 4 (harmful if swallowed) according to the GHS (EU).

- Category 1 for eye irritation or serious eye damage according to GHS (EU).

1. Acute / short-term exposure - systemic effects

Cutaneous route

No mortality and no local changes were noted at a limit dose of 2000 mg/kg in the studies used for assessment by weight of evidence (Bisesi, 1994; Moreno, 1973). The dermal LD50 is therefore greater than 2000 mg/kg and MeS is not classified as hazardous via the dermal route.

No short-term systemic DNEL needs to be derived for cutaneous exposure.

Inhalation

A subacute inhalation toxicity study (Gage, 1970) using MeS vapour at an effectively saturated concentration of 700 mg/m3 for 7 hours per day for 4 weeks did not demonstrate any adverse effects. MeS therefore has low potential for systemic toxicity by inhalation.

A short-term systemic DNEL has been derived for inhalation exposure in case peak exposure is relevant.

Dose Descriptor:

NOAEC_(systemic)rat = 700 mg/m3 (for 7h exposure)

Modification of Dose Descriptor

The modified Haber's law was used to correct the NOAEC from 7 hours to 15 minute exposure. A default value of n=3 was used for extrapolating from longer to shorter exposure duration:

NOAEC_{(15 min)}= 2126 mg/m3

Conversion of an inhalatory rats NOAEC into a corrected inhalatory NAEC for workers:

NAEC_corrected= NOAEC_{inhalatory 15 min}×6.7 m³/10 m³

NAEC_corrected= 1424 mg/m3

6.7 m3/person: respiratory volume without activity for general population ;

10 m3/person: respiratory volume light activity for workers (8h exposure)

Assessment factor (AF):

Interspecies factor (allometric scaling for metabolic rate) 1 (not applicable)

Intraspecies factor (default value for worker variability) 5

Exposure duration: 1 (adjusted above)

Overall assessment factor (OAF): 5

DNEL= NAEC_(corrected)/OAF = 1424 mg/m3 / 5= 285 mg/m3

DNEL_{(acute inhalation local)}= 285 mg/m3

2. Acute / short-term exposure - local effects

Irritation and corrosion

Skin irritation

In the key study (Sentient company study), MeS did not elicit any skin reactions. MeS is not classified as a skin irritant.

No short-term local DNEL needs to be derived for cutaneous exposure.

Eye irritation

MeS is classified as “Category 1” for eye irritation or serious eye damage according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) criteria

No short-term local DNEL needs to be derived (qualitative assessment: moderate hasard).

Inhalation

The subacute inhalation toxicity study (Gage, 1970) using MeS vapour at an effectively saturated concentration of 700 mg/m3 for 7 hours per day for 4 weeks did not demonstrate any local effects. MeS therefore has low potential for local effects by inhalation.

No short-term local DNEL needs to be derived for inhalation exposure.

Sensitization

The skin sensitization potential of MeS has been examined in many studies in animals in several test systems. All mazimization studies in guinea pigs, the majority of studies by other protocols in guinea pigs and the majority of LLNA studies in mice give a non-sensitizing result.Overall, the weight of evidence from both animal tests and human data suggests that MeS may have, at most, weak skin sensitization potential.

Since MeS is not classified as a skin sensitizer it is not relevant to derive a DNEL for this endpoint.

3. Long-term exposure - systemic effects

Cutaneous route

Since MeS may be absorbed by the skin, it is relevant to derive a DNEL for long term exposure by cutaneous route even though it is not classified as hazardous by cutaneous exposure.

The only available dermal toxicity study was conducted at very high doses, was limited in design and in reporting detail and is not useful for risk assessment.

A route to route extrapolation has been carried out (oral-to-dermal) based on an oral long term study (Webb, 1965). MeS was administered to rats at dietary concentrations of 0, 0.1%, 0.5%, 1.0% or 2.0% (equivalent to 0, 50, 250, 500, and 1000 mg/kg bw/day) for two years. Body weight of both sexes were significantly decreased in both the 1.0% and 2.0% groups and an increased amount of cancellous bone was present in the metaphyses in rats treated at 1.0% or 2.0%, with minimal increase for one rat at 0.5%.

The NOAEL was 0.1% (50 mg/kg bw/day). The LOAEL was 0.5% (250 mg/kg bw/day) for body weight gain reduction and bone lesions in rats.

Dose Descriptor:

NOAEL rat (oral) = 50 mg/kg bw/day

Modification of Dose Descriptor

Route-to-route: In mice, MeS was absorbed 100% by oral route against 40% by dermal route (Yamagata, 1976).

Dermal NAEL_(corrected)= oral NOAEL x 100 / 40 =125 mg/kg bw/day.

Assessment factor (AF):

Interspecies factor (allometric scaling for metabolic rate) 4 (rat)

Interspecies factor (remaining differences) 1 (NOAEL rat & dog similar)

Intraspecies factor (default value for worker variability) 5

Exposure duration: 1 (chronic study)

Dose-response: 1 (NOAEL)

Overall assessment factor (OAF): 20

DNEL= NAEL_(corrected)/OAF = 125 mg/kg / 20= 6.25 mg/kg/day

DNEL_{(long-term dermal systemic)}= 6 mg/kg/day

Inhalation

One subacute inhalation toxicity study on MeS at a single limit dose level (saturated vapour) and chronic oral studies in rats and dogs are available for risk assessment.

Assessment based on subacute inhalation study

Four rats were exposed to a saturated atmosphere (700 mg/m3) of MeS for 7 hours per day, 5 days per week for 4 weeks. MeS did not cause any adverse effects (Gage, 1970).

Dose Descriptor:

NOAEC rat = 700 mg/m3 (for sub-acute exposure).

Modification of Dose Descriptor

Conversion of an inhalatory rat NOAEC into a corrected inhalatory NAEC for workers:

NAEC_corrected= NOAEC_inhalatory= 700 x 7h/8h× 6.7 m3/10 m3

NAEC_corrected= 410.4 mg/m3

6.7 m3/person: respiratory volume without activity for general population;

10 m3/person: respiratory volume light activity for workers (8h exposure)

7h:time exposure of rats in Gage study

8h:time exposure of workers

Assessment factor (AF):

Interspecies factor (allometric scaling for metabolic rate) 1 (not applicable)

Interspecies factor (remaining differences) 1 (NOAEL rat & dog similar)

Intraspecies factor (default value for worker variability) 5

Exposure duration: 6 (subacute to chronic study)

Dose-response: 1 (NOAEL)

Overall assessment factor (OAF): 30

DNEL= NAEC_(corrected)/OAF = 410.4 mg/m3 / 30= 13.68 mg/kg/day

DNEL_{(long-term inhalation systemic)}=14 mg/m3

Assessment based on chronic dietary rat study

A route to route extrapolation has been carried out (oral-to-inhalation) based on an oral long term study performed on MeS (Webb, 1965). MeS was administered to rats at dietary concentrations of 0, 0.1%, 0.5%, 1.0% or 2.0% (equivalent to 0, 50, 250, 500, and 1000 mg/kg bw/day) for two years. Body weight of both sexes were significantly decreased in both the 1.0% and 2.0% groups and an increased amount of cancellous bone was present in the metaphyses in rats treated at 1.0% or 2.0%, with minimal increase for one rat at 0.5%.

The NOAEL was 0.1% (50 mg/kg bw/day). The LOAEL was 0.5% (250 mg/kg bw/day) for body weight gain reduction and bone lesions in rats.

Dose Descriptor:

NOAEL rat (oral) = 50 mg/kg bw/day

Modification of Dose Descriptor

Route-to-route: Since MeS is readily absorbed by both oral and dermal routes, no correction was required.

Conversion of an oral rat NOAEL into a corrected inhalatory NAEC for workers:

NAEC_corrected= NOAEL_oral50 mg/kg x 70 kg/10 m³

NAEC_corrected= 350 mg/m3

70 kg person

10 m3/person: respiratory volume light activity for workers (8h exposure)

Assessment factor (AF):

Interspecies factor (allometric scaling for metabolic rate) 4 (rat)

Interspecies factor (remaining differences) 1 (NOAEL rat & dog similar)

Intraspecies factor (default value for worker variability) 5

Exposure duration: 1 (chronic study)

Dose-response: 1 (NOAEL)

Overall assessment factor (OAF): 20

DNEL= NAEC_(corrected)/OAF = 350 mg/m3 / 20= 17.5 mg/m3

DNEL_{(long-term inhalation systemic)}=17.5 mg/m3

The DNELs calculated from the subchronic inhalation study at limit dose (saturated vapour) and the oral study are therefore consistent and that derived from the longer (chronic) study has been taken for risk assessment.

DNEL_{(long-term inhalation systemic)}=17.5 mg/m3

Carcinogenicity

The chronic study (Webb, 1965) described above was selected as a key study. No carcinogenic effect was observed. MeS is not considered as carcinogenic.

Since MeS is not considered as carcinogenic, no DNEL or DMEL should be derived for this endpoint.

Reprotoxicity

Fertility

The key study for the potential of MeS to impair fertility 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. The NOAEL (parental/reproduction) determined in this study was 250 mg/kg bw/day.

Developmental Toxicity

No reliable studies on the effect of MeS on development are available.

This endpoint has been assessed on basis of a 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. A read-across justification is provided as attachment to IUCLID section 13 respectively as appendix to the CSR.

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.

FDA published a report mentioning developmental toxicity data in rabbit and in rat, but they refered to unpublished reports that are not accessible to the registrants. Several actions were launched in order to obtain an access to the original reports, but no agreement has been reached at the moment. The critical study is however reported in a short summary taken from the FDA report and ECHA CLH report, but with a reliability of 4 and not useable for the hasard and risk assessment.

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.

Since, MeS is not considered as reprotoxic for humans, no DNEL should be derived for this endpoint.

4. Long-term exposure - local effects

Cutaneous route

No data on repeated cutaneous exposure are available.

Since MeS is not classified as irritating to the skin, no long-term local DNEL needs to be derived for cutaneous exposure.

Inhalation

Since MeS is not classified as irritating to the skin, respiratory system, no long-term local DNEL needs to be derived for inhalation exposure.

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

4 mg/m³

Most sensitive endpoint:

repeated dose toxicity

Overall assessment factor (AF):

40

Modified dose descriptor starting point:

NOAEC

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

213 mg/m³

Most sensitive endpoint:

repeated dose toxicity

Overall assessment factor (AF):

10

Modified dose descriptor starting point:

NOAEC

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

3 mg/kg bw/day

Most sensitive endpoint:

repeated dose toxicity

Modified dose descriptor starting point:

NOAEL

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

no hazard identified

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

1 mg/kg bw/day

Most sensitive endpoint:

repeated dose toxicity

Overall assessment factor (AF):

40

Modified dose descriptor starting point:

NOAEL

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

5 mg/kg bw/day

Most sensitive endpoint:

repeated dose toxicity

Overall assessment factor (AF):

40

Modified dose descriptor starting point:

NOAEL

Hazard assessment conclusion:

medium hazard (no threshold derived)

Introduction

In this section, data from all end-points are examined and analyzed in order to determine for which it is relevant and possible to establish a DNEL value. The method followed is that proposed in the guidance for the implementation of REACH (Chapter R.8: Characterisation of dose (concentration)-response for human health, May 2008).

Self Classification:

- Methyl salicylate is self-classified:

- Category 4 (harmful if swallowed) according to the GHS (EU).

-Category 1 for eye irritation or serious eye damage according to GHS (EU).

1. Acute / short-term exposure - systemic effects

Cutaneous route

No mortality and no local changes were noted at a limit dose of 2000 mg/kg in the studies used for assessment by weight of evidence (Bisesi, 1994; Moreno, 1973). The dermal LD50 is therefore greater than 2000 mg/kg and MeS is not classified as hazardous via the dermal route.

No short-term systemic DNEL needs to be derived for cutaneous exposure.

Inhalation

A subacute inhalation toxicity study (Gage, 1970) using MeS vapour at an effectively saturated concentration of 700 mg/m3 for 7 hours per day for 4 weeks did not demonstrate any adverse effects. MeS therefore has low potential for systemic toxicity by inhalation.

A short-term systemic DNEL has been derived for inhalation exposure in case peak exposure is relevant.

Dose Descriptor:

NOAEC_(systemic)rat = 700 mg/m3 (for 7h exposure)

Modification of Dose Descriptor

The modified Haber's law was used to correct the NOAEC from 7 hours to 15 minute exposure. A default value of n=3 was used for extrapolating from longer to shorter exposure duration:

NOAEC_{(15 min)}= 2126 mg/m3

No correction is required for ventilatory rate:

NAEC_corrected= 2126 mg/m3

Assessment factor (AF):

Interspecies factor (allometric scaling for metabolic rate) 1 (not applicable)

Intraspecies factor (default value for general population variability) 10

Exposure duration: 1 (adjusted above)

Overall assessment factor (OAF): 10

DNEL= NAEC_(corrected)/OAF = 2126 mg/m3 / 10= 212.6 mg/m3

DNEL_{(acute inhalation local)}= 213 mg/m3

Oral route

MeS is classified as harmful if swallowed and may be used at low concentrations as a flavouring ingredient. Adverse effects from single high doses have been reported. It is therefore relevant to derive a DNEL for acute term exposure by the oral route.

DNEL calculation is based on a set of subchronic (6-12-week) oral studies (Abbott & Harrison, 1978). MeS was administered to rats at dietary concentrations of 0.2%, 0.36%, 0.63%, 1.13%, or 2.0% (equivalent to 100, 180, 320, 560 and 1000 mg/kg/day ) MeS. Bone lesions and growth retardation were observed in rats fed MeS at 1% and 2% in diet.

The NOAEL was 0.36% (180 mg/kg bw/day).

Dose Descriptor:

NOAEL rat (oral) = 180 mg/kg bw/day

Modification of Dose Descriptor

No modification of dose descriptor is required.

Oral NAEL_(corrected)= oral NOAEL x 1 =180 mg/kg bw/day.

Assessment factor (AF):

Interspecies factor (allometric scaling for metabolic rate) 4 (rat)

Interspecies factor (remaining differences) 1 (NOAEL rat & dog similar)

Intraspecies factor (default value for general population variability) 10

Exposure duration: 1 (subchronic study used for acute)

Dose-response: 1 (NOAEL)

Overall assessment factor (OAF): 40

DNEL= NAEL_(corrected)/OAF = 180 mg/kg / 40= 4.5 mg/kg/day

DNEL_{(acute oral systemic)}= 5 mg/kg/day

2. Acute / short-term exposure - local effects

Irritation and corrosion

Skin irritation

In the key study (Sentient, date), MeS did not elicit any skin reactions. MeS is not classified as a skin irritant.

No short-term local DNEL needs to be derived for cutaneous exposure.

Eye irritation

MeS is classified as “Category 1” for eye irritation or serious eye damage according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) criteria

No short-term local DNEL needs to be derived (qualitative assessment: moderate hasard).

Inhalation

The subacute inhalation toxicity study (Gage, 1970) using MeS vapour at an effectively saturated concentration of 700 mg/m3 for 7 hours per day for 4 weeks did not demonstrate any local effects. MeS therefore has low potential for local effects by inhalation.

No short-term local DNEL needs to be derived for inhalation exposure.

Sensitization

The skin sensitization potential of MeS has been examined in many studies in animals in several test systems. All mazimization studies in guinea pigs, the majority of studies by other protocols in guinea pigs and the majority of LLNA studies in mice give a non-sensitizing result.Overall, the weight of evidence from both animal tests and human data suggests that MeS may have, at most, weak skin sensitization potential.

Since MeS is not classified as a skin sensitizer it is not relevant to derive a DNEL for this endpoint.

3. Long-term exposure - systemic effects

Cutaneous route

Since MeS may be absorbed by the skin, it is relevant to derive a DNEL for long term exposure by cutaneous route even though it is not classified as hazardous by cutaneous exposure.

The only available dermal toxicity study was conducted at very high doses, was limited in design and in reporting detail and is not useful for risk assessment.

A route to route extrapolation has been carried out (oral-to-dermal) based on an oral long term study (Webb, 1965). MeS was administered to rats at dietary concentrations of 0, 0.1%, 0.5%, 1.0% or 2.0% (equivalent to 0, 50, 250, 500, and 1000 mg/kg bw/day) for two years. Body weight of both sexes were significantly decreased in both the 1.0% and 2.0% groups and an increased amount of cancellous bone was present in the metaphyses in rats treated at 1.0% or 2.0%, with minimal increase for one rat at 0.5%.

The NOAEL was 0.1% (50 mg/kg bw/day. The LOAEL was 0.5% (250 mg/kg bw/day) for body weight gain reduction and bone lesions in rats.

Dose Descriptor:

NOAEL rat (oral) = 50 mg/kg bw/day

Modification of Dose Descriptor

Route-to-route: Since MeS is readily absorbed by both oral and dermal routes, no correction was required.

Dermal NAEL_(corrected)= oral NOAEL x 1 =50 mg/kg bw/day.

Assessment factor (AF):

Interspecies factor (allometric scaling for metabolic rate) 4 (rat)

Interspecies factor (remaining differences) 1 (NOAEL rat & dog similar)

Intraspecies factor (default value for worker variability) 5

Exposure duration: 1 (chronic study)

Dose-response: 1 (NOAEL)

Overall assessment factor (OAF): 20

DNEL= NAEL_(corrected)/OAF = 50 mg/kg / 20= 2.5 mg/kg/day

DNEL_{(long-term dermal systemic)}= 3 mg/kg/day

Inhalation

One subacute inhalation toxicity study on MeS at a single dose level and chronic oral studies in rats and dogs are available for risk assessment.

Assessment based on subacute inhalation study

Four rats were exposed to a saturated atmosphere (700 mg/m3) of MeS for 7 hours per day, 5 days per week for 4 weeks. MeS did not cause any adverse effects (Gage, 1970).

Dose Descriptor:

NOAEC rat = 700 mg/m3 (for sub-acute exposure).

Modification of Dose Descriptor

Conversion of an inhalatory rat NOAEC into a corrected inhalatory NAEC for general population:

NAEC_corrected= NOAEC_inhalatory700 x 7h/24h

NAEC_corrected= 204.2 mg/m3

7h:time exposure of rats in Gage study

24h:worst case time exposure of general population

Assessment factor (AF):

Interspecies factor (allometric scaling for metabolic rate) 1 (not applicable)

Interspecies factor (remaining differences) 1 (NOAEL rat & dog similar)

Intraspecies factor (default value for general population variability) 10

Exposure duration: 6 (subacute to chronic study)

Dose-response: 1 (NOAEL)

Overall assessment factor (OAF): 60

DNEL= NAEC_(corrected)/OAF = 204.2 mg/m3 / 60= 3.4 mg/kg/day

DNEL_{(long-term inhalation systemic)}= 3 mg/m3

Assessment based on chronic dietary rat study

A route to route extrapolation has been carried out (oral-to-inhalation) based on an oral long term study performed on MeS (Webb, 1965). MeS was administered to rats at dietary concentrations of 0, 0.1%, 0.5%, 1.0% or 2.0% (equivalent to 0, 50, 250, 500, and 1000 mg/kg bw/day) for two years. Body weight of both sexes were significantly decreased in both the 1.0% and 2.0% groups and an increased amount of cancellous bone was present in the metaphyses in rats treated at 1.0% or 2.0%, with minimal increase for one rat at 0.5%.

The NOAEL was 0.1% (50 mg/kg bw/day). The LOAEL was 0.5% (250 mg/kg bw/day) for body weight gain reduction and bone lesions in rats.

Dose Descriptor:

NOAEL rat (oral) = 50 mg/kg bw/day

Modification of Dose Descriptor

Route-to-route: Since MeS is readily absorbed by both oral and dermal routes, no correction was required.

Conversion of an oral rat NOAEL into a corrected inhalatory NAEC for general population:

NAEC_corrected= NOAEL_oral50 mg/kg x 70 kg/20 m³

NAEC_corrected= 175 mg/m3

70 kg person

20 m3/person: respiratory volume general population (worst case for 24h exposure)

Assessment factor (AF):

Interspecies factor (allometric scaling for metabolic rate) 4 (rat)

Interspecies factor (remaining differences) 1 (NOAEL rat & dog similar)

Intraspecies factor (default value for general population variability) 10

Exposure duration: 1 (chronic study)

Dose-response: 1 (NOAEL)

Overall assessment factor (OAF): 40

DNEL= NAEC_(corrected)/OAF = 175 mg/m3 / 40= 4.38 mg/m3

DNEL_{(long-term inhalation systemic)}= 4 mg/m3

The DNELs calculated from the subchronic inhalation study at limit dose and the oral study are therefore consistent and that derived from the longer (chronic) study has been taken for risk assessment.

DNEL_{(long-term inhalation systemic)}= 4 mg/m3

Oral route

MeS is classified as harmful if swallowed and may be used at low concentrations as a flavouring ingredient, it is relevant to derive a DNEL for long term exposure by the oral route.

DNEL calculation is based on an oral long term study (Webb, 1965). MeS was administered to rats at dietary concentrations of 0, 0.1%, 0.5%, 1.0% or 2.0% (equivalent to 0, 50, 250, 500, and 1000 mg/kg bw/day) for two years. Body weight of both sexes were significantly decreased in both the 1.0% and 2.0% groups and an increased amount of cancellous bone was present in the metaphyses in rats treated at 1.0% or 2.0%, with minimal increase for one rat at 0.5%.

The NOAEL was 0.1% (50 mg/kg bw/day. The LOAEL was 0.5% (250 mg/kg bw/day) for body weight gain reduction and bone lesions in rats.

Dose Descriptor:

NOAEL rat (oral) = 50 mg/kg bw/day

Modification of Dose Descriptor

No modification of dose descriptor is required.

Oral NAEL_(corrected)= oral NOAEL x 1 =50 mg/kg bw/day.

Assessment factor (AF):

Interspecies factor (allometric scaling for metabolic rate) 4 (rat)

Interspecies factor (remaining differences) 1 (NOAEL rat & dog similar)

Intraspecies factor (default value for general population variability) 10

Exposure duration: 1 (chronic study)

Dose-response: 1 (NOAEL)

Overall assessment factor (OAF): 40

DNEL= NAEL_(corrected)/OAF = 50 mg/kg / 40= 1.25 mg/kg/day

DNEL_{(long-term oral systemic)}= 1 mg/kg/day

Carcinogenicity

The chronic study (Webb, 1965) described above was selected as a key study. No carcinogenic effect was observed. MeS is not considered as carcinogenic.

Since MeS is not considered as carcinogenic, no DNEL or DMEL should be derived for this endpoint.

Reprotoxicity

Fertility

The key study for the potential of MeS to impair fertility 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. The NOAEL (parental/reproduction) determined in this study was 250 mg/kg bw/day.

Developmental Toxicity

No reliable studies on the effect of MeS on development are available. This endpoint has been assessed on basis of a 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. A read-across justification is provided as attachment to IUCLID section 13 respectively as appendix to the CSR.

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.

FDA published a report mentioning developmental toxicity data in rabbit and in rat, but they refered to unpublished reports that are not accessible to the registrants. Several actions were launched in order to obtain an access to the original reports, but no agreement has been reached at the moment. The critical study is however reported in a short summary taken from the FDA report and ECHA CLH report, but with a reliability of 4 and not useable for the hasard and risk assessment.

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.

Since MeS is not considered as reprotoxic for humans, no DNEL should be derived for this endpoint

4. Long-term exposure - local effects

Cutaneous route

No data on repeated cutaneous exposure are available.

Since MeS is not classified as irritating to the skin, no long-term local DNEL needs to be derived for cutaneous exposure.

Inhalation

Since MeS is not classified as irritating to the skin, respiratory system or eyes, no long-term local DNEL needs to be derived for inhalation exposure.

Registration Dossier

Toxicological Summary

Administrative data

Workers - Hazard via inhalation route

Systemic effects

Long term exposure

DNEL related information

Acute/short term exposure

DNEL related information

Local effects

Long term exposure

Acute/short term exposure

DNEL related information

Workers - Hazard via dermal route

Systemic effects

Long term exposure

DNEL related information

Acute/short term exposure

DNEL related information

Local effects

Long term exposure

Acute/short term exposure

Workers - Hazard for the eyes

Local effects

Additional information - workers

General Population - Hazard via inhalation route

Systemic effects

Long term exposure

DNEL related information

Acute/short term exposure

DNEL related information

Local effects

Long term exposure

Acute/short term exposure

DNEL related information

General Population - Hazard via dermal route

Systemic effects

Long term exposure

DNEL related information

Acute/short term exposure

DNEL related information

Local effects

Long term exposure

Acute/short term exposure

General Population - Hazard via oral route

Systemic effects

Long term exposure

DNEL related information

Acute/short term exposure

DNEL related information

General Population - Hazard for the eyes

Local effects

Additional information - General Population