Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate

Endpoint summary

Description of key information

Acute oral toxicity: Key study: OECD 423 / EU method B.1 tris, GLP study. The LD50 of the test item is higher than 2000 mg/kg body weight by oral route in the rat. The LD50 cut-off of the test item may be considered to be higher than 5000 mg/kg body weight by oral route in the rat.

Acute inhalation toxicity: Data waiving (study scientifically not necessary). The substance is considered to be inappropriate as exposure of humans via inhalation is unlikely taking into account the vapour pressure of the substance (0.0778 Pa at 20°C) and/or the possibility of exposure to aerosols, particles or droplets of an inhalable size.

Acute dermal toxicity:Key study: EU Method B.3, GLP study. The LD50 value in an acute dermal toxicity study (on structual analogue, Cyclohexyl salicylate) with rabbits was LD50 > 2000 mg/kg bw and the substance is not classified for acute dermal toxicity according to the CLP regulation

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 9th, 2018 to May 29th, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

December 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: JANVIER LABS (53940 Legenest St. Isle - France)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8-9 weeks old
- Weight at study initiation: 213 ± 16.3 g
- Fasting period before study: overnight.
- Housing: Groups of three in solid-bottomed clear polycarbonate cages with a stainless steel mesh lid. Each cage contains sawdust bedding which was changed at least 2 times a week. Each cage was installed in conventional air-conditioned animal husbandry.
- Diet (e.g. ad libitum): Foodstuff (ENVIGO -2016) ad libitum.
- Water (e.g. ad libitum): Tap-water from public distribution system ad libitum. Microbiological and chemical analyses of the water were carried out once every six months by Bureau Veritas- Eurofins (France).
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 ºC
- Humidity (%): 30-70%
- Air changes (per hr): 10 changes/hour
- Photoperiod (hrs dark / hrs light): 12 h light (7 - 19 h) / 12 h darkness

IN-LIFE DATES: From March 7th, 2018 To May 23rd, 2018

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

MAXIMUM DOSE VOLUME APPLIED: 2.39 mL/kg (test item), 10 mL/kg (control).
CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: no indication of toxicity based on available data. Therefore, the selected starting dose was 2000 mg/kg body weight.

Doses:

2000 mg/kg

No. of animals per sex per dose:

6

Control animals:

yes

Remarks:

Study No. TAO423-2018-001 (see "Other information on results").

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Systemic observations at 30 min, 1h, 3h, 4h, 24h, 48h
after administration and daily for 14 days. Weighing: on day 0 (just before administering the test item) then on Day 2, Day 7, and Day 14.
- Necropsy of survivors performed: yes. At termination, macroscopic observations were performed. Macroscopic observations were entered on individual autopsy sheets. Only those organs likely to be modified in cases of acute toxicity were examined. Parameters examined: Oesophagus, Stomach, Duodenum, Jejunum, Ileum, Caecum, Colon, Rectum, Spleen, Liver, Thymus, Trachea, Lungs, Heart, Kidneys, Urinary Bladder, Ovaries, Uterus, Treatment Area, Adrenals and Pancreas.
- Other examinations performed: Clinical signs: Spontaneous activity, Preyer's reflex (noise), Respiratory rate, Convulsions, Tremors, Body temperature, Muscle tone, Palpebral opening, Pupil appearance, Salivation, Lachrymation, Righting reflex, Back hair appearance, Mortality.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality occurred during the study.

Clinical signs:

other: A decrease of spontaneous activity (6/6), muscle tone (5/6) and righting reflex (5/6) was noted a t30 minutes post dose. The animals recovered normal activity at 24 hours post dose.

Gross pathology:

The macroscopic examination of the animals at the end of the study did not reveal treatment-related changes.

Table 1. Test item at 2000 mg/kg bw. Body weight and weight gain in grams.

FEMALES	D0	D2	D2-D0	D7	D7-D0	D14	D14-D0
Rf 2488 Rf 2489 Rf 2490	191 200 196	225 220 210	24 20 14	238 249 247	47 49 51	250 270 265	59 70 69
Rf 2532 Rf 2533 Rf 2534	224 236 211	243 250 228	19 14 17	256 267 238	32 31 27	271 282 249	47 46 38
MEAN	209.7	229.3	19.7	249.2	39.5	264.5	54.8
Standard deviation	17.5	14.8	7.4	11.1	10.6	12.9	13.2

Table 2. Clinical signs

OBSERVATIONS:	FEMALES			FEMALES
T0 + 30 minutes	Rf 2488	Rf 2489	Rf 2490	Rf 2532	Rf 2533	Rf 2534
Spontaneous activity	D	D	D	D	D	D
Preyer’s reflex (noise)	N	N	N	N	N	N
Respiratory rate	N	N	N	N	N	N
Convulsions	N	N	N	N	N	N
Tremors	N	N	N	N	N	N
Body temperature	N	N	N	N	N	N
Muscle tone	D	D	D	N	N	N
Palpebral opening	N	N	N	N	N	N
Pupil appearance	N	N	N	N	N	N
Salivation	N	N	N	N	N	N
Lachrymation	N	N	N	N	N	N
Righting reflex	D	D	D	N	N	N
Back hair appearance	N	N	N	N	N	N
MORTALITY	0	0	0	0	0	0
Remarks	None			None

OBSERVATIONS:	FEMALES			FEMALES
T0 + 1 hour	Rf 2488	Rf 2489	Rf 2490	Rf 2532	Rf 2533	Rf 2534
Spontaneous activity	D	D	D	N	D	D
Preyer’s reflex (noise)	N	N	N	N	N	N
Respiratory rate	N	N	N	N	N	N
Convulsions	N	N	N	N	N	N
Tremors	N	N	N	N	N	N
Body temperature	N	N	N	N	N	N
Muscle tone	N	N	N	N	N	N
Palpebral opening	N	N	N	N	N	N
Pupil appearance	N	N	N	N	N	N
Salivation	N	N	N	N	N	N
Lachrymation	N	N	N	N	N	N
Righting reflex	N	N	N	N	N	N
Back hair appearance	N	N	N	N	N	N
MORTALITY	0	0	0	0	0	0
Remarks	None			None

OBSERVATIONS:	FEMALES			FEMALES
T0 + 3 hours T0 + 4 hours	Rf 2488	Rf 2489	Rf 2490	Rf 2532	Rf 2533	Rf2534
Spontaneous activity	D	D	D	N	D	D
Preyer’s reflex (noise)	N	N	N	N	N	N
Respiratory rate	N	N	N	N	N	N
Convulsions	N	N	N	N	N	N
Tremors	N	N	N	N	N	N
Body temperature	N	N	N	N	N	N
Muscle tone	N	N	N	N	D	D
Palpebral opening	N	N	N	N	N	N
Pupil appearance	N	N	N	N	N	N
Salivation	N	N	N	N	N	N
Lachrymation	N	N	N	N	N	N
Righting reflex	N	N	N	N	D	D
Back hair appearance	N	N	N	N	N	N
MORTALITY	0	0	0	0	0	0
Remarks	None			None

OBSERVATIONS:	FEMALES			FEMALES
D1 to D14	Rf 2488	Rf 2489	Rf 2490	Rf 2532	Rf 2533	Rf 2534
Spontaneous activity	N	N	N	N	N	N
Preyer’s reflex (noise)	N	N	N	N	N	N
Respiratory rate	N	N	N	N	N	N
Convulsions	N	N	N	N	N	N
Tremors	N	N	N	N	N	N
Body temperature	N	N	N	N	N	N
Muscle tone	N	N	N	N	N	N
Palpebral opening	N	N	N	N	N	N
Pupil appearance	N	N	N	N	N	N
Salivation	N	N	N	N	N	N
Lachrymation	N	N	N	N	N	N
Righting reflex	N	N	N	N	N	N
Back hair appearance	N	N	N	N	N	N
MORTALITY	0	0	0	0	0	0
Remarks	None			None

Table 3. Necropsy findings

Found dead:  GENERAL APPEARANCE		Euthanasia BEFORE AUTOPSY:Normal		X	At	term	X
			Observed Organs	Observations
* SOPHAGUS * STOMACH * DUODENUM * JEJUNUM * ILEON * CAECUM * COLON * RECTUM * SPLEEN * LIVER			X X X X X X X X XX	N.t.R. N.t.R. N.t.R. N.t.R. N.t.R. N.t.R. N.t.R. N.t.R. N.t.R. N.t.R.
* THYMUS			X	N.t.R.
* TRACHEA * LUNGS * HEART			XXX	N.t.R. N.t.R. N.t.R.
* KIDNEYS * URINARYBLADDER * OVARIES * UTERUS			X X X X	N.t.R. N.t.R. N.t.R. N.t.R.
* TREATMENT AREA			-	-
* ADRENALS * PANCREAS			XX	N.t.R. N.t.R.
PARTICULARS: None

Found dead:  GENERAL APPEARANCE		Euthanasia: BEFORE AUTOPSY:Normal		X	At	term	X
			Observed Organs	Observations
* ESOPHAGUS * STOMACH * DUODENUM * JEJUNUM * ILEON * CAECUM * COLON * RECTUM * SPLEEN * LIVER			X X X X X X X X XX	N.t.R. N.t.R. N.t.R. N.t.R. N.t.R. N.t.R. N.t.R. N.t.R. N.t.R. N.t.R.
* THYMUS			X	N.t.R.
* TRACHEA * LUNGS * HEART			X X X	N.t.R. N.t.R. N.t.R.
* KIDNEYS * URINARYBLADDER * OVARIES * UTERUS			X X X X	N.t.R. N.t.R. N.t.R. N.t.R.
* TREATMENT AREA			-	-
* ADRENALS * PANCREAS			XX	N.t.R. N.t.R.
PARTICULARS: None

Interpretation of results:

GHS criteria not met

Remarks:

EU Criteria

Conclusions:

The LD50 of the test item is higher than 2000 mg/kg body weight by oral route in the rat. The LD50 cut-off of the test item may be considered to be higher than 5000 mg/kg body weight by oral route in the rat.

Executive summary:

The acute oral toxicity of the test item has been tested in accordance with OECD 423 and EU B.1 tris, under GLP conditions. A limit test was performed, where the test item was administered to a total of 6 female Sprague-Dawley rats (class method) at the dose of 2000 mg/kg body weight by oral gavage. All animals were observed immediately after administration for the onset of any toxic signs and once daily thereafter for 14 days. No mortality occurred during the study and the body weight evolution of the animals remained normal during the study. The decrease of spontaneous activity (6/6), muscle tone (5/6) and righting reflex (5/6) was noted a t30 minutes post dose. The animals recovered normal activity at 24 hours post dose. Macroscopic examination of the animals at the end of the study did not reveal treatment related changes. No other signs of systemic toxicity were noted. The LD50 of the test item is higher than 2000 mg/kg body weight by oral route in the rat. The LD50 cut-off of the test item may be considered to be higher than 5000 mg/kg body weight by oral route in the rat.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

5 000 mg/kg bw

Quality of whole database:

Klimish score 1

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because exposure of humans via inhalation is not likely taking into account the vapour pressure of the substance and/or the possibility of exposure to aerosols, particles or droplets of an inhalable size

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
According to REACH annex VIII column 2 the study does not need to be conducted if:

It is considered to be inappropriate as exposure of humans via inhalation is unlikely taking into account the vapour pressure of the substance (0.0778 Pa at 20°C) and/or the possibility of exposure to aerosols, particles or droplets of an inhalable size.

Clinical signs:

other:

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study in accordance with EU Method B.3 To address toxicological endpoints as part of the REACH registration of Amyl Salicylate (Target Substance) it is proposed to read-across to Cyclohexyl Salicylate (Source Substance). The use of read-across works within the spirit of REACH and the stated aim of the legislation to reduce animal testing where possible. The Target Substance and Source Substance have been characterised using the categories and databases present in the OECD [Q]SAR Toolbox. From the profiling, it can be seen that the two substances share structural similarities and also ‘mechanistic action’ similarities which are both general and endpoint specific. Therefore read across is justified.

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rabbit

Strain:

other: "Kleinrusse" Chbb: HM

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Karl Thomae GmbH, Biberach
- Age at study initiation: young adult
- Weight at study initiation: 2286 g (males), 2156 g (females)
- Housing: individually in rabbit batteries
- Diet (e.g. ad libitum): Altromin rabbit food 2023 ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: female animals were acclimatised for four days, male animals were maintained at the laboratory for about 2 months

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 21
- Humidity (%): 45 to 50
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

Type of coverage:

occlusive

Vehicle:

other: aqueous suspension with 2% carboxymethyl cellulose and 0.5% Cremophor

Details on dermal exposure:

TEST SITE
- Area of exposure: back and on the sides
- % coverage: about 10% of skin surface area
- Type of wrap if used: application area was covered by gauze and polyethylene foil that was fixed with Leukosilk strips; the whole area was then wrapped with an elastic bandange (Acrylastik Kompressionsbinde) with acrylate adhesive

REMOVAL OF TEST SUBSTANCE
- Washing (if done): washed-off with water
- Time after start of exposure: after removal of cover following 24 hours after application

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): between 8.4 and 9.5 g of the preparation containing the test substance at a level of 50% was applied to the skin of the animals by brushing the preparation onto the skin
VEHICLE
- Amount(s) applied (volume or weight with unit): the substance was applied as an aqueous solution containing 2% carboxymethylcellulose and 0.5% Cremophor

Duration of exposure:

24 hours

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5 females, 5 males

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations: Directly and 1, 2, 3, 4 and 6 hours after application, then twice daily
- Frequency of weighing: animals were weighed one hour before application and 1, 7 and 14 days after application
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight,organ weights, histopathology, other: skin reaction

Statistics:

not applicable

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality occurred.

Clinical signs:

other: Slightly reduced activity during the first six hours after application; slightly reduced body temperature in three females and one male during first six hours after application

Gross pathology:

No findings were reported.

Other findings:

Skin reactions: Slight reddening of skin after application (erythema score of 1 according to Draize); clear signs of erythema in all animals after removal of coverage; signs of erythema were reversible within 5 days, but scaling was observed until test day 12

Interpretation of results:

GHS criteria not met

Conclusions:

The LD50 value in an acute dermal toxicity study with rabbits was LD50 > 2000 mg/kg bw and the substance is not classified for acute dermal toxicity according to the CLP regulation.

Executive summary:

The acute toxicity of the substance Cyclohexyl salicylate was studied under GLP in accordance with the EU Method B.3. Five female and five male young adult rabbits of the strain "Kleinrusse" were used in the test. Females and males had body weights of 2156 g and 2286 g, respectively. The substance was applied to the shave skin of the back and on the sides (application area about 10% of the total skin surface area) in form of a preparation, which was an aqueous suspension containing 50% of the test substance, 2% carboxymethylcellulose and 0.5% Cremophor. The preparation was brushed onto the skin and animals were then exposed to the material under occlusion for 24 hours. After this exposure period, the coverage was removed and remaining test substance was washed-off with water. None of the animals died after application or during the 14-day observation period. A slight body weight reduction was observed after application. Animals exhibited signs of skin irritation (erythema persisted for up to 5 days, scaling occured and persisted until test day 12). No clinical signs were observed after patch removal. No pathological findings were made at necropsy following the 14-day observation period. The acute dermal toxicity study resulted in a LD50 value > 2000 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Klimish score 2

Additional information

Acute oral toxicity:

Key study: The acute oral toxicity of the test item has been tested in accordance with OECD 423 and EU method B.1 tris, following GLP. The test item was administered to a total of 6 female Sprague-Dawley rats at the dose of 2000 mg/kg body weight by oral gavage (class method). All animals were observed immediately after administration for the onset of any toxic signs and once daily thereafter for 14 days. No mortality occurred during the study and the body weight evolution of the animals remained normal during the study. A decrease of spontaneous activity (6/6), muscle tone (5/6) and righting reflex (5/6) was noted at t30 minutes postdose. The animals recovered normal activity at 24 hours post-dose. Macroscopic examination of the animals at the end of the study did not reveal treatment-related changes. No other signs of systemic toxicity were noted. The LD50 of the test item is higher than 2000 mg/kg body weight by oral route in the rat. The LD50 cut-off of the test item may be considered to be higher than 5000 mg/kg body weight by oral route in the rat.

Acute dermal toxicity:

The acute toxicity of the substance Cyclohexyl salicylate was studied under GLP in accordance with the EU Method B.3. Five female and five male young adult rabbits of the strain "Kleinrusse" were used in the test. Females and males had body weights of 2156 g and 2286 g, respectively. The substance was applied to the shave skin of the back and on the sides (application area about 10% of the total skin surface area) in form of a preparation, which was an aqueous suspension containing 50% of the test substance, 2% carboxymethylcellulose and 0.5% Cremophor. The preparation was brushed onto the skin and animals were then exposed to the material under occlusion for 24 hours. After this exposure period, the coverage was removed and remaining test substance was washed-off with water. None of the animals died after application or during the 14-day observation period. A slight body weight reduction was observed after application. Animals exhibited signs of skin irritation (erythema persisted for up to 5 days, scaling occured and persisted until test day 12). No clinical signs were observed after patch removal. No pathological findings were made at necropsy following the 14-day observation period. The acute dermal toxicity study resulted in a LD50 value > 2000 mg/kg bw.

Justification for classification or non-classification

Based on the available information (oral LD50 5000 mg/kg bw, dermal LD50>2000 mg/kg bw), the substance is not classified for acute toxicity according to CLP Regulation (EC) no. 1272/2008.