2-hydroxybenzoic acid 2-butyloctyl ester

Toxicity to reproduction

Administrative data

Endpoint:

three-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Justification for type of information:

To address some of the toxicological endpoints as part of the REACH registration of 2-Hydroxybenzoic acid, 2-Butyloctyl ester (CAS 190085-41-7) (target substance), it is proposed to read across to methyl salicylate (CAS 119-36-8) (source substance) to fulfill the data requirements of the OECD 416 study.
Both the above mentioned substance share similar chemical structures. As mentioned, both substances have identical functionality, they are both esters of salicylic acid. The only difference in the two substances is in the alcohol portion of the ester group.
Read across has been deemed as suitable.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

review article or handbook

Title:

Evaluation of safety for food additives: An illustration involving the influence of methyl salicylate on rat reproduction

Author:

Gross MA, Fitzhugh OG

Year:

1970

Bibliographic source:

Biometrics 26:181-184

Reference 2

Reference Type:

publication

Title:

Effect of methyl salicylate on rat reproduction

Author:

Collins TFX, Hansen WH, Keeler HV

Year:

1971

Bibliographic source:

Toxicol. Appl. Pharmacol. 18:755-765

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

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

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methyl salicylate

EC Number:

204-317-7

EC Name:

Methyl salicylate

Cas Number:

119-36-8

IUPAC Name:

methyl salicylate

Details on test material:

- Name of test material: methyl salicylate
- Molecular formula: C8H8O3
- Molecular weight: 152
- Substance type: oily liquid
- Physical state: no data
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Purity test date: no data
- Lot/batch No.: no data
- Stability under test conditions: no data
- Storage condition of test material: no data
- Source: Dodge and Olcott, Inc., New York

Test animals

Species:

rat

Strain:

Osborne-Mendel

Sex:

male/female

Details on test animals or test system and environmental conditions:

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


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


IN-LIFE DATES: no data

Administration / exposure

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

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

no information available

Duration of treatment / exposure:

100 days prior to the first mating until study termination

Frequency of treatment:

continuos in the feed

Details on study schedule:

no information available

Doses / concentrations

Remarks:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

no information available

Positive control:

none employed

Examinations

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 examined

Statistics:

The Chi-square 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/number of females mated).

Offspring viability indices:

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

- Fertility index: no significant differences for any dose in the 1st generation. Appreciable decreases seen in the 2nd and 3rd generations at the highest dose level (5000 ppm).
- Average litter size/female: significant decreases were seen in the second generation in the second mating at 3000 ppm and in both matings 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.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reduced neonate viability

Remarks on result:

other: Generation: development

2

Dose descriptor:

NOAEL

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reduced neonate viability

Remarks on result:

other: Generation: development

3

Dose descriptor:

NOAEL

Effect level:

1 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant effects at the highest dose tested

Remarks on result:

other: Generation: reproduction

4

Dose descriptor:

NOAEL

Effect level:

1 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant effects at the highest dose tested

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

VIABILITY (OFFSPRING)

- The average number of liveborn young per mated female: 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. of 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 the 0, 3000 and 5000 ppm dose levels showed no indication of toxic effects.

Effect levels (F1)

1

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

2

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 500 ppm (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

body weight and weight gain

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

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

 

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

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

(a) Fertility Index (number of litters/ number of females mated)

(b) Percent females pregnant

 

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

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

(a) Total number progeny/number females mated

(b) Average litter size per female mated

(c) significant at P<0.01

(d) significant at P<0.05

 

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

dietary level (ppm)
		0			500			1500			3000			5000
gen.	mating	No. (a)	Av. (b)	VI (c, d)	No.	Av.	VI (c, d)	No.	Av.	VI (c, d)	No.	Av.	VI (c, d)	No.	Av.	VI (c, d)
1	1	208/20	10.4	1,00	211/19	11.1	1,00	195/20	9,8	0,94	229/20	11,4	0,97	167/18	9,3	0,88
	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 mated

(b) Average number liveborn per female mated

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

(d) Not analyzed for statistical significance

(e) significant at P<0.05

(f) significant at P<0.01

 

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

dietary level (ppm)
		0			500			1500			3000			5000
gen.	mating	No. (a)	Av. (b)	SI (c, d)	No.	Av.	SI	No.	Av.	SI	No.	Av.	SI	No.	Av.	SI
1	1	157/17	9,2	0,90	116/14	8,3	0,82	172/19	9,1	0,96	152/15	10,1	0,92	129/15	8,6	0,94
	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 mated

(b) Average number day 4 survivors per female mated

(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

 

See "Overall remarks, attachments" for Table 5.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, methyl salicylate did not significantly reduce male or female fertility. Methyl salicylate did induce developmental toxicity: adverse effects on offspring viability were observed but with no evidence of increased incidence of malformations at any dose 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 this 3 -generation study, 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 methyl salicylate, or 22.5, 67.5, 135, 225 mg/kg bw as salicylic acid) 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 indexes at any dose level at the F1 generation, however it was considered that there were "appreciable" decreases at 250 mg/kg in the F2 and F3 generations. 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 the F2 generation. No external malformations were reported in pups of any litter and necropsy of the third generation weanlings showed no significant findings. The effects in a calcium carbonate supplement groups did not differ significantly from those of the groups fed methyl salicylate alone.