Sodium anisate

Endpoint summary

Description of key information

short-term repeated dose toxicity OECD 422, oral, rat: systemic NOAEL 500 mg/kg bw/day (highest dose tested)

Read-across from anisaldehyde (CAS 123-11-5)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Refer to analogue justification provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

reference to same study

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

12/13 males and 11/13 females of the high dose group showed transient salivation.

Mortality:

no mortality observed

Description (incidence):

No dead or moribund animals were seen in any sex and treatment group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Males: slightly increased (104%) at both 100, 500 mg/kg bw/day compared to the vehicle control values (not statistically significant) at the end of the study;
- Females:
(pre-)mating: slightly increased body weights (107% at day 14) at 500 mg/kg bw/day compared to the vehicle control values (not statistically significant);
gestation: significantly increased body weights on days 0 and 7 of gestation (113% and 108% of control respectively) at 500 mg/kg bw/day; marked individual differences not reaching statisitcal significance after day 14 of gestation in this dose group;
lactation: significantly increased body weights on day 0 of lactation at 100 and 500 mg/kg bw/day (108% and 115% of control respectively), slight increase on day 4 of lactation (104% of control) at 500 mg/kg bw/day (not statistically significant).
See Tables 1 and 2 for data on male and female body weight development.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Males: significantly increased only on day 35 at 100 mg/kg bw/day; significantly increased from day 7 onward at 500 mg/kg bw/day
- Females: significantly increased during the premating period (days 7 and 13) at 500 mg/kg bw/day

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

platelet count: significant decrease in males at 500 mg/kg bw/day and in females at 100 and 500 mg/kg bw/day (no effects were noted on prothrombin time or activated partial thromboplastin time)
red blood cell count: significant decrease in males at 100 and 500 mg/kg bw/day
mean corpuscular volume: significant increases in females at 100 mg/kg bw/day
mean corpuscular hemoglobin: significant increases in females at 100 mg/kg bw/day

No significant changes in hematocrit value and hemoglobin content (males/females), reticulocyte count and the histopathological examination of bone marrow; spleen did not show any changes related to hematological changes. Therefore, all the changes in the erythrocyte were considered by the authors to occur accidentally.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A/G ratio: significant increases in males at 500 mg/kg bw/day
A/G ratio: significant decreases in females at 100 mg/kg bw/day
GOT activity: significant increases in males at 500 mg/kg bw/day
Inorganic phosphorus: significant increases in males at 500 mg/kg bw/day
Albumin: significant decreases in females at 100 mg/kg bw/day
Chloride: significant increases in females at 100 mg/kg bw/day
Glucose: significant increase in females at 500 mg/kg bw/day
Calcium: significant decrease in females at 500 mg/kg bw/day

Females changes (A/G ratio, albumin, chloride) were not dose-dependent and therefore considered not to be test substance related.
Male changes (A/G ratio, inorganic phosphorus) were - although statistically significant - considered to be of no toxicological relevance because their were only marginal in magnitude, not observed in females or changes noted in females were reciprocal in nature.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver: increase in absolute (113%; statistically non-significant) and relative (by 111%; statistically significant) weight in males at 500 mg/kg bw/day
Liver: increase in absolute (117%; statistically significant) and relative (109%; statistically non-significant) weight in females at 500 mg/kg bw/day
Epididymis: statistically significant decreases in absolute/relative (91%/88%) weight in males at 500 mg/kg bw/day

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related findings were noted at 500 and 100 mg/kg bw/day (dose-dependent incidence):
500 mg/kg bw/day
Males: 1 animal with black spots in the glandular stomach mucosa
Females: 3 animals with dark or black spots/areas in glandular stomach; 1 animal with black deposits in glandular stomach

Females in 100 mg/kg bw/day
2 animals with thickening of the forestomach mucosa, one of which was accompanied by edema, the other had black spots in the glandular stomach mucosa.

Incidental findings affecting only single treated animals or noted in the control group:
500 mg/kg bw/day
Males: 1 animal with epididymal nodule
Females: 1 animal with cystic ovarian bursa

100 mg/kg bw/day
Single animal findings (dark-red spot in the lung; ovarian cyst on the right side; accumulated white gelatinous content in the vagina cavity)

Females in 20 mg/kg bw/day
Single animal findings (darkening of the caudate lobe of the liver, diverticulum in the ileum, abnormal formation of kidney)

Males and females in the control group:
Single animal findings (1 female with small thymus; 1 male with dark-red spot in the lung)

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related findings were noted at 500 and 100 mg/kg bw/day (dose-dependent incidence):
- Stomach: squamous cell hyperplasia in the forestomach of all animals at 500 mg/kg bw/day and also in the forestomach for 3 (male)/5 (female) animals at 100 mg/kg bw/day.

- Liver: all males/3 females with very slight - slight centrilobular hypertrophy of hepatocytes (500 mg/kg bw/day).

Incidental findings affecting only single treated animals or noted in the control group:

Stomach: in female animals, very slight-slight submucosal forestomach edema in 1/1/5/3 animals (0/20/100/500 mg/kg bw/day). Single female (controls) and 2 females (100 mg/kg bw/day) with very slight-slight lymphocytic infiltration in the glandular stomach mucosa. One male (20 mg/kg bw/day) with very slight lymphocytic and neutrophile infiltrations in the muscular layer.

Liver: almost all animals in control and treated groups with very slight-moderate periportal fatty changes (13/13/13/12 males and 12/11/13/13 females at 0/20/100/500 mg/kg bw/day, respectively).

- Testes: single animal findings (very slight granulation tissue in the seminiferous tubule interstitium in 1 animal at 500 mg/kg bw/day; 1 animal of control and 1 animal of 100 mg/kg bw/d showed very slight atrophy in the seminiferous tubule). None of these abnormalities was deemed to cause a failure of conception.

- Epididymes: single animal findings (slight unilateral sperm granuloma) at 100 mg/kg bw/day.
High dose and control group animals showed comparable interstitial lymphocytic infiltration in the prostate. No abnormalities were seen in the seminal vesicle.

- Kidney: 2 females and 1 male (500 mg/kg bw/d) with very slight -moderate basophilic tubules in the cortex (vs. 4 males and 1 female in controls); 1 of these females with slight vacuolar degeneration in the proximal renal tubule.

- Heart (only males): very slight - slight myocardial degeneration/fibrosis (9/10/8/12 males at 0/20/100/500 mg/kg bw/day, respectively). There were no differences in the onset frequency and severity between the control group and each test substance-treated group.

- Spleen: All male and female animals (control; 500 mg/kg bw/day) with very slight - slight extramedullary hematopoiesis and brown pigmentation. No differences in the frequency and severity between the two groups observed.

- Pancreas: Single animal finding (male 500 mg/kg bw/day) with very slight focal acinar cell atrophy.

- Vagina, Ovary and Uterus: 1 animal (control) with cyst in the lamina propria; no other abnormalities observed.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

other: the findings reported for the rat are considered not to be relevant for humans

Remarks on result:

other:

Remarks:

source: CAS 123-11-5, Hatano Institute, 2010, rat

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no systemic adverse effects noted up to and including the high dose of 500 mg/kg bw/day; adaptative, non-adverse effects seen in the liver at 500 mg/kg bw/day

Remarks on result:

other:

Remarks:

source: CAS 123-11-5, Hatano Institute, 2010, rat

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (actual dose received)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Table 1. Body weight changes in males

	Body weight gain (g, mean ± S.D.)
Dose level (mg/kg bw/day)	0	20	100	500
N	13	13	13	13
Day of administration
1 (initial body weight)	381.8 ± 12.0	385.1± 13.2	383.2± 13.5	384.4± 13.4
7	406.3 ± 17.1	407.9± 20.3	413.8± 21 1	413.1± 21.3
14	431.7 ± 24.1	434.7 ± 20.3	440.4 ± 28.3	442.9 ± 27.4
21	451.8 ± 27.2	458.0 ± 21.8	467.8 ± 30.9	469.1 ± 32.5
28	480.4 ± 33.8	490.2 ± 25.6	498.9 ± 33.9	499.5 ± 35.6
35	407.9 ± 38.5	518.7 ± 31.6	527.6 ± 39.4	529.0 ± 42.2
42	525.8 ± 44.6	537.5 ± 38.7	547.4 ± 44.1	549.2 ± 45.8

Table 2. Body weight changes in females

	Body weight gain (g, mean ± S.D. (N)
Dose level (mg/kg bw/day)	0	20	100	500
Day of administration
1	236.5 ± 8.4 (13)	237.4 ± 10.8 (13)	237.0 ± 9.6 (13)	237.9 ± 9.8 (13)
7	247.1 ± 10.9 (13)	251.2 ± 12.4 (13)	252.1 ± 13.1 (13)	257.5 ± 16.2 (13)
14	254.5 ± 13.0 (13)	259.6 ± 15.4 (13)	260.4 ± 16.4 (13)	271.4 ± 19.4 (13)
21				303.3 (1)
Day of pregnancy
0	262.1 ± 14.3 (12)	269.2 ± 16.0 (12)	271.6 ± 13.0 (12)	249.9 ± 15.2 **(6)
7	302.4 ± 11.7 (12)	307.7 ± 16.5 (12)	309.7 ± 16.3 (12)	325.8 ± 19.3 * (6)
14	340.7 ± 14.8 (12)	346.8 ± 15.8 (12)	349.0 ± 18.9 (12)	359.9 ± 29.6 (6)
20	412.2 ± 31.7 (12)	420.4 ± 15.5 (12)	414.1 ± 30.3 (12)	416.6 ± 61.9 (6)
Day of lactation
0	306.7 ± 14.7	317.7 ± 20.2	331.5 ± 21.9 **	352.3 ± 14.1 * (5)
4	337.4 ± 16.9	335.7 ± 12.5	335.7 ± 15.9	352.1 ± 11.7 (5)

*  significantly different from control, p < 0.05

** significantly different from control, p < 0.01

Table 3. Selected haematological findings

Males (N = 13)
Dose level (mg/kg bw/day)	RBC (x10^4/mm^3)	Hemoglobin (g/dL)	Hematocrit (%)	MCV (µm^3)	MCH (pg)	Platelet (x10^4/mm^3)	PT (sec)	APTT (sec)
0	811 ± 35	15.2 ± 0.6	44.6 ± 1.8	55.0 ± 1.9	18.8 ± 0.6	91.6 ± 7.4	14.6 ± 1.5	20.9 ± 1.6
20	801 ± 33	15.2 ± 0.5	44.3 ± 2.1	55.3 ± 1.3	19.0 ± 0.5	94.7 ± 11.5	14.5 ± 1.4	21.4 ± 2.3
100	771 ± 33**	14.8 ± 0.8	43.1 ± 1.9	55.9 ± 1.9	19.2 ± 0.6	86.2 ± 7.7	14.4 ± 1.1	21.4 ± 1.5
500	779 ± 23*	14.9 ± 0.4	43.3 ± 1.5	55.6 ± 1.7	19.1 ± 0.6	82.4 ± 8.6*	13.8 ± 1.0	19.7 ± 1.9
Females (N = 13)
0	684 ± 48	13.3 ±0.8	39.8 ± 2.4	58.2 ± 1.5	19.4 ± 0.6	108.6 ± 11.5	13.0 ± 0.5	17.9 ± 1.2
20	649 ± 44	13.0± 0.8	38.8 ± 2.0	58.8 ± 2.2	20.0 ± 0.6	111.2 ± 9.3	12.7 ± 0.6	17.1 ± 1.5
100	649 ± 67	13.2 ± 1.3	39.1 ± 4.0	60.3 ± 2.3*	20.3 ± 0.7**	96.6 ± 11.8*	12.5 ± 0.6	16.9 ± 0.9
500	712 ± 69	14.0 ± 1.1	41.5 ± 3.5	58.4 ± 1.9	19.7 ±0.7	87.3 ± 8.9**	12.9 ± 0.7	16.8 ± 0.8

*  significantly different from control, p < 0.05

** significantly different from control, p < 0.01

Table 4. Selected clinical biochemistry findings

Males (N = 13)
Dose level (mg/kg bw/day)	Total protein (g/dL)	Albumin (g/dL)	A/G	ALP (U/L)	GPT (U/L)	GOT (U/L)	Inorg. Phosph. (mg/dL)
0	5.5 ± 0.3	3.0 ± 0.2	1.23 ± 0.19	211 ± 68	30 ± 4	62 ± 7	6.3 ± 0.3
20	5.5± 0.2	3.1 ± 0.2	1.37 ± 0.12	243 ± 47	27 ± 4	57 ± 5	6.5 ± 0.5
100	5.3± 0.3	3.0 ± 0.2	1.30 ± 0.14	210 ± 44	31 ± 5	34 ± 5	6.2 ± 0.4
500	5.2± 0.2	3.1 ± 0.2	1.42 ± 0.16**	248 ± 86	39 ± 16	84 ± 46*	6.7 ± 0.7*
Females (N = 13)
0	5.5 ± 0.4	3.2 ± 0.3	1.44 ± 0.16	113 ± 22	42 ± 15	77 ± 18	6.3 ± 0.9
20	5.5 ± 0.4	3.1 ± 0.2	1.37 ± 0.18	123 ± 98	38 ± 8	68 ± 9	6.1 ± 0.6
100	5.3 ± 0.3	2.9 ± 0.2*	1.24 ± 0.08**	125 ± 34	40 ± 12	74 ± 16	6.0 ± 0.8
500	5.2 ± 0.2	3.1 ± 0.2	1.54 ± 0.20	124 ± 35	67 ± 26	67 ± 26	6.1 ± 0.6

*  significantly different from control, p < 0.05

** significantly different from control, p < 0.01

Conclusions:

The Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Screening Test (OECD 422) with the source substance anisaldehyde (CAS 123-11-5) was was considered for read-across as key study to cover the endpoint of repeated dose toxicity.
The read across approach is justified in the analogue justification. The target and source substances are considered unlikely to differ in their repeated dose toxicity potential.

In this oral OECD 422 study in rats, doses levels of 20, 100 and 500 mg/kg bw/day anisaldehyde were tested. No adverse systemic findings were noted in this OECD 422 study in rats. The noted findings on local irritation of the forestomach are not considered relevant for humans. Therefore, a NOAEL for repeated dose toxicity after oral exposure was set at 500 mg/kg bw/day and is also considered relevant for the target substance sodium anisate (CAS 536-45-8).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 1) from a surrogate substance with similar structure and intrinsic properties. Read-across is justified based on (bio)transformation to common compound(s) (refer to endpoint discussion for further details). The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VII, 8.5, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

In the absence of any evidence for species specific effects or modes of action (beside changes in the forestomach) the effects observed in animals and the absence of effects are regarded as relevant for humans.

Additional information

Justification for read-across

There are no data available on repeated dose toxicity of sodium anisate (CAS 536-45-8) or on p-anisic acid (CAS 100-09-4). In order to fulfil the standard information requirements set out in Annex VIII - IX, 8.6, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from a structurally related substance was conducted. In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.”In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across) “to avoid the need to test every substance for every endpoint”. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID section 13).

 

Repeated dose toxicity: oral

A reliable oral Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test with anisaldehyde (CAS 123-11-5) is available and was performed according to OECD 422 (Hatano Research Institute, 2000) and in compliance with GLP. Groups of 13 male and 13 female Sprague-Dawley rats were exposed to the test substance at dose levels of 20, 100 and 500 mg/kg bw/day by oral gavage once daily for 7 days/week for at least 42 (males) and 40 (females) consecutive days. Control animals (13 per sex and dose) received the concurrent vehicle, corn oil, only. Observations and examinations of the animals included clinical signs, body weight, food consumption, haematology, clinical chemistry, urinalysis, organ weights, gross necropsy, organ weights and histopathology. No mortality was observed and except for transient salivation noted in most high dose animals no clinical signs were noted. Body weights and food consumption was marginally increased in mid and high dose males and in high dose females. These findings were not considered adverse. Platelet count was slightly although statistically significantly decreased in high dose males and mid and high dose females. In the absence of any effects on coagulation times this finding was considered to be of no toxicological relevance. Red blood cell count was significantly decreased in mid and high dose males. However, as no significant changes in hematocrit value and hemoglobin content, reticulocyte count and the histopathological examination of bone marrow were seen and the spleen did not show any changes related to hematological changes, these changes were considered to have occurred incidentally. Absolute and relative liver weights were increased in high dose males and females. However, histopathology revealed only very slight to slight centrilobular hypertrophy of hepatocytes, which is considered an adaptive rather than an adverse effect. During necropsy black spots or black deposits in the glandular stomach as well as thickening of the forestomach mucosa were noted in a few high dose males and mid and high dose females. Histopathology revealed no changes in the glandular stomach but squamous cell hyperplasia in the forestomach of all high dose animals and also in the forestomach for 3 male and 5 female mid dose animals. In the absence of adverse systemic effects, a NOAEL of 500 mg/kg bw/day for systemic toxicity was derived, whereas a NOAEL of 20 mg/kg bw/day for local effects was derived based on stomach irritation noted at 100 mg/kg bw/day and above. Histopathological stomach findings were limited to the forestomach, an organ that has no human correlate. Therefore, these effects were considered to have no relevance for humans and do not warrant a classification of the source substance. Accordingly, based on read across, also classification of the the target substance is not warranted.

Justification for classification or non-classification

The available data on short-term repeated dose toxicity as available from an OECD 422 study conducted with the source substance anisaldehyde (CAS 123-11-5) do not indicate adverse effects up to and including 500 mg/kg bw/day (the highest dose tested). Therefore, no adverse effects are expected for the target substance at doses up to and including 500 mg/kg bw/day. Therefore and based on read across, classification of sodium anisate with respect to repeated dose exposure is not warranted according to Regulation (EC) No. 1272/2008.