Registration Dossier

This review of acetic acid for REACh has not uncovered any information to undermine the following statement.
“Long-term toxicity/carcinogenicity studies in animals with oral exposure are not necessary, considering that humans are exposed to orally ingested acetic acid from various food sources and there is no evidence that such exposure is causally related to toxic effects and an increased cancer incidence. Therefore, no new animal toxicity studies conforming to long-term toxicity or carcinogenicity test guidelines are necessary for Annex I inclusion of acetic acid.” (EU DAR, 2008).

Endpoint conclusion:

no study available

Endpoint conclusion:

no study available

Endpoint conclusion:

no study available

Reference

Endpoint:

chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non- GLP, non- guideline study, published in peer reviewed literature. No restrictions, fully adequate for assessment.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline available

Principles of method if other than guideline:

Acetic acid was used as a promotor for tumour development in mice initiated with DMBA or β-PL and was applied dermally 1-3 times per week (at doses of 1-40 mg/animal) for 32 weeks. Control animals received acetic acid dermally once per week. The incidence of papillomas and carcinomas was recorded and they were removed at random for histological verification.

GLP compliance:

no

Limit test:

no

Species:

mouse

Strain:

CD-1

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Mouse Farms, North Wilmington, Massachusetts, USA
- Age at study initiation: 7-12 weeks old
- Weight at study initiation: not reported
- Housing: not reported
- Diet (e.g. ad libitum): not reported
- Water (e.g. ad libitum): not reported

Type of coverage:

not specified

Vehicle:

acetone

Details on exposure:

TEST SITE
- Area of exposure: No details reported
- % coverage: No details reported
- Type of wrap if used: No details reported
- Time intervals for shavings or clippings: shaved 2 days before exposure

REMOVAL OF TEST SUBSTANCE
- no details reported

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.2 mL in acetone
- Concentration (if solution): 33, 167, 333, 500, 667, 833 or 1000 μmoles/animal
- Constant volume or concentration used: yes

VEHICLE
- Justification for use and choice of vehicle (if other than water): No details reported
- Amount(s) applied (volume or weight with unit): 0.2 mL

USE OF RESTRAINERS FOR PREVENTING INGESTION: no details

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

32 weeks

Frequency of treatment:

1-3 times per week.
Controls dosed once per week.

Remarks:

Doses / Concentrations:
0, 1. 2, 10, 20, 40 mg/animal
Basis:

Remarks:

Doses / Concentrations:
0, 17, 33, 167, 333, 667 μmoles
Basis:

No. of animals per sex per dose:

20-30

Control animals:

yes, sham-exposed

Positive control:

0.25% croton oil

Observations and examinations performed and frequency:

The animals were observed and the incidence of papillomas and carcinomas was recorded weekly

Sacrifice and pathology:

GROSS PATHOLOGY: No data
HISTOPATHOLOGY: Yes - during the 32 week study, papillomas and carcinomas were removed for histological verification at random

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Details on results:

Mortality:
- A single dermal application of acetic acid at doses of up to 40 mg/animal, in mice initiated with ß-PL or DMBA did not induce excessive mortality. However, more than one weekly application of 10-40 mg acetic acid caused excessive mortality. 33% of mice died when 10 mg acetic acid/animal was applied dermally 3 times per week and approximately 50% of mice died when 20 mg was applied twice a week.

Dose descriptor:

NOAEL

Remarks:

toxicity

Effect level:

30 other: mg/animal

Sex:

female

Basis for effect level:

other: No deaths when applied dermally once per week for 32 weeks

Dose descriptor:

LOAEL

Remarks:

toxicity

Effect level:

10 other: mg/animal

Sex:

female

Basis for effect level:

other: Mortality (33%) when applied dermally 3 times per week.

Critical effects observed:

not specified

Executive summary:

Acetic acid was used as a promotor for tumour development in mice initiated with DMBA or β-PL and was applied dermally 1-3 times per week (at doses of 1-40 mg/animal) for 32 weeks. Control animals received acetic acid dermally once per week.

A single dermal application of acetic acid at doses of up to 40 mg/animal, in mice initiated with ß-PL or DMBA did not induce excessive mortality. However, more than one weekly application of 10 -40 mg acetic acid caused excessive mortality. 33% of mice died when 10 mg acetic acid/animal was applied dermally 3 times per week and approximately 50% of mice died when 20 mg was applied twice a week.

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

Reliable with restrictions (Klimisch score=2).

Reference

Endpoint:

chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non- GLP, non- guideline study, published in peer reviewed literature. No restrictions, fully adequate for assessment.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline available

Principles of method if other than guideline:

Acetic acid was used as a promotor for tumour development in mice initiated with DMBA or β-PL and was applied dermally 1-3 times per week (at doses of 1-40 mg/animal) for 32 weeks. Control animals received acetic acid dermally once per week. The incidence of papillomas and carcinomas was recorded and they were removed at random for histological verification.

GLP compliance:

no

Limit test:

no

Species:

mouse

Strain:

CD-1

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Mouse Farms, North Wilmington, Massachusetts, USA
- Age at study initiation: 7-12 weeks old
- Weight at study initiation: not reported
- Housing: not reported
- Diet (e.g. ad libitum): not reported
- Water (e.g. ad libitum): not reported

Type of coverage:

not specified

Vehicle:

acetone

Details on exposure:

TEST SITE
- Area of exposure: No details reported
- % coverage: No details reported
- Type of wrap if used: No details reported
- Time intervals for shavings or clippings: shaved 2 days before exposure

REMOVAL OF TEST SUBSTANCE
- no details reported

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.2 mL in acetone
- Concentration (if solution): 33, 167, 333, 500, 667, 833 or 1000 μmoles/animal
- Constant volume or concentration used: yes

VEHICLE
- Justification for use and choice of vehicle (if other than water): No details reported
- Amount(s) applied (volume or weight with unit): 0.2 mL

USE OF RESTRAINERS FOR PREVENTING INGESTION: no details

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

32 weeks

Frequency of treatment:

1-3 times per week.
Controls dosed once per week.

Remarks:

Doses / Concentrations:
0, 1. 2, 10, 20, 40 mg/animal
Basis:

Remarks:

Doses / Concentrations:
0, 17, 33, 167, 333, 667 μmoles
Basis:

No. of animals per sex per dose:

20-30

Control animals:

yes, sham-exposed

Positive control:

0.25% croton oil

Observations and examinations performed and frequency:

The animals were observed and the incidence of papillomas and carcinomas was recorded weekly

Sacrifice and pathology:

GROSS PATHOLOGY: No data
HISTOPATHOLOGY: Yes - during the 32 week study, papillomas and carcinomas were removed for histological verification at random

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Details on results:

Mortality:
- A single dermal application of acetic acid at doses of up to 40 mg/animal, in mice initiated with ß-PL or DMBA did not induce excessive mortality. However, more than one weekly application of 10-40 mg acetic acid caused excessive mortality. 33% of mice died when 10 mg acetic acid/animal was applied dermally 3 times per week and approximately 50% of mice died when 20 mg was applied twice a week.

Dose descriptor:

NOAEL

Remarks:

toxicity

Effect level:

30 other: mg/animal

Sex:

female

Basis for effect level:

other: No deaths when applied dermally once per week for 32 weeks

Dose descriptor:

LOAEL

Remarks:

toxicity

Effect level:

10 other: mg/animal

Sex:

female

Basis for effect level:

other: Mortality (33%) when applied dermally 3 times per week.

Critical effects observed:

not specified

Executive summary:

Acetic acid was used as a promotor for tumour development in mice initiated with DMBA or β-PL and was applied dermally 1-3 times per week (at doses of 1-40 mg/animal) for 32 weeks. Control animals received acetic acid dermally once per week.

A single dermal application of acetic acid at doses of up to 40 mg/animal, in mice initiated with ß-PL or DMBA did not induce excessive mortality. However, more than one weekly application of 10 -40 mg acetic acid caused excessive mortality. 33% of mice died when 10 mg acetic acid/animal was applied dermally 3 times per week and approximately 50% of mice died when 20 mg was applied twice a week.

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

30

Study duration:

subchronic

Quality of whole database:

Reliable with restrictions (Klimisch score=2).
NOAEL (toxicity): 30 mg/animal (female) (No deaths when applied dermally once per week for 32 weeks).
LOAEL 10 mg/mouse glacial acetic acid , applied dermally 3 times per week., (Mortality (33%) induced skin irritation and associated hyperplasia

Acetic acid, as an active substance, has been considered under the fourth stage of the re-evaluation programme of the Plant Protection Product Directive 91/414/EEC (provisions of Regulation (EC) No 1112/2002) for use as a herbicide on e. g. pome and stonefruit, ornamental shrubs, paths. The decision has been made that it is included in Annex I of that Directive (Commission Directive 2008/127/EC of 18 December 2008 amending Council Directive 91/414/EEC to include several active substances). The use of acetic acid as a Plant Protection Product is therefore regarded as registered under REACh (Article 15 of EC Reg 1907/2006). The toxicology endpoints addressed under Dir. 91/414/EEC to conclude on toxicological hazard are directly comparable with those under REACh.

EFSA supported the recommendations by the rapporteur Member State and the result of the examination in accordance with the provisions of Article 24a of Regulation 2229/2004 stating that there are clear indications that it may be expected that this use of acetic acid does not result in any harmful effects on human health (EFSA, 2013). The Draft Assessment Report (EU DAR, 2008) states that “Long-term toxicity/carcinogenicity studies in animals with oral exposure are not necessary, considering that humans are exposed to orally ingested acetic acid from various food sources and there is no evidence that such exposure is causally related to toxic effects and an increased cancer incidence. Therefore, no new animal toxicity studies conforming to long-term toxicity or carcinogenicity test guidelines are necessary for Annex I inclusion of acetic acid.”

Acetic acetic was more recently reviewed as a feed additive (EFSA, 2012). Acetic acid, calcium acetate and sodium diacetate are listed in the EU Register of Feed Additives as preservatives for use with feed for all animal species without restrictions. There is now an application for the re-authorisation of acetic acid and these salts as preservatives in feed and for a new use of acetic acid as preservative in water for drinking. They may be used alone or in combination with other organic acids typically in a concentration 200 to 2,500 mg acetate/kg complete feedingstuffs. Acetic acid and its salts are considered equivalent when compared on a molar basis. Based on the comparison between consumer and target animals exposure to acetic acid and the limited experimental data available for chickens and the dogs fed diets containing acetate, a maximum concentration of 2,500 mg acetic acid/kg complete feed (or 1000 mg/L water for drinking) is considered safe for poultry, pigs and pet animals. The tolerance of ruminants is considerably higher. No data is available for salmonids. Given the complete and rapid metabolism of acetate, the use of acetate in animal nutrition is not expected to contribute to human exposure. Acetic acid and its salts are bulk industrial chemicals and the hazards for those handling these substances are well known and documented. Acetates are normal components of the diet of humans and animals and are produced in molar quantities in the gastrointestinal tract. They are fully metabolised and so do not pose a risk to the environment. Acetic and its salts have the potential to act as preservatives in feedingstuffs and water for drinking (EFSA 2012).

EFSA also published aScientific Opinion on the substantiation of health claims related to acetic acid and maintenance of normal blood pressure (ID 1447) pursuant to Article 13(1) of Regulation (EC) No 1924/20061 (EFSA 2011) in which they concluded “In weighing the evidence, the Panel took into account that although one animal study showed an effect of acetic acid administration on systolic blood pressure, results from two human intervention studies are conflicting, and that a sustained effect of orally administered acetic acid on blood pressure is unlikely because of its rapid absorption and clearance from the circulation after consumption.”

In addition to consideration of the status under EC Dir. 91/414/EEC, it is also approved for use as a food additive by the Joint FAO/WHO Expert Committee on Food Additives (Rome, 11-18 October, 1966). In the USA acetic acid is affirmed as Generally Recognised as Safe (GRAS) by the US FDA (21CFR184.1005) and has also been exempted from the requirement of a tolerance under the US EPA Federal Food, Drug, and Cosmetic Act (FFDCA).

Although for some endpoints modern, guideline studies are not available, below is the discussion of the studies reviewed for this submission and no new information has been sourced which conflicts the conclusions of EFSA (EFSA, 2013).

Non-human

Information from chronic exposure studies on acetic acid is available (Kondo et al., 2001, Lamb and Evard, 1919 and Slaga et al., 1988). Whilst these studies are not conventional toxicology studies the information provided by them aids understanding of the properties of acetic acid.

In the first study, groups of 6 spontaneously hypertensive rats were fed diets containing 6% (w/w) acetic acid, 6% (w/w) rice vinegar or control diet for 8 weeks (Kondo et al., 2001). Blood pressure, heart rate, body weight, food intake and water consumption were measured at weekly intervals. Urine samples were collected every 2 weeks for measurements of volume, sodium, calcium and catecholamine excretion. After 8 weeks, animals were killed and blood samples collected from the aorta. The heart, aorta, kidneys and lungs were removed and the angiotensin I-converting enzyme (ACE) activity was measured.

No changes in bodyweight gain or clinical signs were observed in any group. Between groups food consumption was similar, with about 290 mg/kg/day of acetic acid being consumed in the acetic acid test group. At an age of about 6 or 7 weeks, a rapid rise in blood pressure to the adult value was noted in all groups. In the groups exposed to acetic acid in diet blood pressure initially rose to the control value (approx 185 mm Hg) before establishing a plateau at the lower value of 160-170 mm Hg towards the end of the 8-week study. There were no adverse effects of acetic acid treatment in SHR rats fed 290 mg/kg/day, the highest level tested.

In an early study, young pigs (from a single litter) were divided into groups of 2 and fed basal diet (control group) or basal diet plus addition of acetic acid (Lamb & Evard, 1919). The dose level was raised every 10-30 days from approximately 155 mg/kg/day to 380-450 mg/kg/day after 60 days. The pigs were weighed during the study and urine samples were taken at intervals to determine ammonia content. At the end of the study blood samples were taken for pH measurement.

There were no mortalities or effects on body weight gain or in early morning urinary ammonia and terminal blood pH between control and test pigs. Acetic acid, at dose levels of up to 450 mg/kg/day, maintained for approximately 6 months, did not affect growth or acid-base balance of pigs.

In another study, topically applied acetic acid was used as a promoter for tumour development in mice that was initiated with beta-propiolactone or dimethylbenzyl[a]anthracene (Slaga et al., 1988). The concentrated acid was applied dermally 1-3 times per week (at doses of 1-40 mg/animal) for 32 weeks. Control animals received acetic acid dermally once per week. The incidence of papillomas and carcinomas was recorded and lesions were removed at random for histological verification.

A single dermal application of acetic acid at doses of up to 40 mg/animal, in mice initiated with beta-propiolactone or dimethylbenzyl[a]anthracene, did not induce excessive mortality. However, more than one weekly application of 10-40 mg acetic acid caused excessive mortality. 33% of mice died when 10 mg acetic acid/animal was applied dermally 3 times per week and approximately 50% of mice died when 20 mg was applied twice a week.

At concentrations above 2 mg/mouse glacial acetic acid, topically applied, induced skin irritation and associated hyperplasia. At 10 mg/mouse, the LOAEL for dermal effects, elevated DNA synthesis was sustained for a period 12-24 hours post-exposure, which is compatible with a mechanism involving cell proliferation (EU DAR, 2008).

Dryden and Hartman (1971) considered the effect on growth of acetate added to diet as the sodium salt (3.58%) (equivalent to 1.58% acetate in the diet (15,800ppm)). They concluded that after a 4 week exposure period to sodium acetate there was no depression of growth.

The results of these studies confirm that for acetic acid the toxicological effect of concern is that of an irritant at the site of first contact. This is consistent with the physiochemical properties of an acid which is capable of producing aqueous solutions of very low pH (25% solution of acetic acid has pH less that 2) and the material is classified under DSD and CLPas corrosive.

Summary of non-human systemic toxicity attributable to acetic acid (oral).

From an oral, drinking water, investigation, a NOAEL of 290 mg/kg/day acetic acid (Kondo et al., 2001; EU DAR, 2008) was reported. This was the highest concentration of acetic acid tested.

Local effects attributable to acetic acid by inhalation or dermal contact are discussed within the irritation section of this dossier.

Human

Acetic acid is a ubiquitous, endogenous substance in humans and animals. The primary route of exposure to exogenous acetic acid for the general population is through consumption of food. Acetic acid occurs naturally in food and is also added to foodstuff as a preservative.

Systemic effects - inhalation exposure

A human volunteer inhalation study (Savina& Anisimov, 1988) has been reported.

Exposure to air concentrations of 5 and 10 mg/m3, for 2 weeks, was not associated with any functional impairments, clinical chemistry or haematological abnormalities. Higher concentrations (15 and 26 mg/m3) were reported to be associated with decreases in ability to concentrate, visual motion activity and intellectual functions (beginning after 3-5 days of exposure), decreased glycolysis in red blood cells (beginning after 1 day of exposure), decrease in physical condition (ergometer performance), increased time required for dark adaptation. Exposure to air concentrations of 5 and 10 mg/m3, with volunteers exposed within chambers with1 hour per day spent at extensive exercise (air inhalation 112 L/min) and the remainder at rest (air inhalation 6 L/min) for 2 weeks was not associated with any functional impairments, clinical chemistry or haematological abnormalities.

In this investigation negative control information was generated only at the very start of each exposure. In contrast, test measurements were reported for individuals continually exposed, within a small chamber, for at least 10 and for up to 22 days. There are no data available for prolonged exposure of these volunteers to air alone. For example, in one experiment under test but not control conditions the temperature was elevated from 21 to 33°C on 3 separate days. Consequently it is difficult to interpret if the changes reported were due to acetic acid exposure or simply due to the prolonged confinement of the individuals within the chamber, or both. In the absence of critical negative control information, this study is unreliable.

Dietary intake - systemic effects

The daily intake of over 100 food additives, including acetic acid, has been estimated for five age groups of Japanese consumers using the shopping basket approach. For acetic acid the intake varied from about 16mg/kg/day for 15 - 19 year olds to 25 mg/kg/day for 1 - 6 year olds, (Ishiwata et al., 2002; see CSR section 5.10.2). Indeed dietary intake excursions to about 240 mg/kg/ day may not be unlikely (EU DAR, 2008). The values compare favourably with the evidence from animal studies indicating that the NOAEL for systemic effects of acetic acid is at least 290 mg/kg /day (highest dose tested).

Systemic and local effects - inhalation exposure

Humans may be exposed to acetic acid via inhalation, oral or dermal routes of exposure. Occupational exposure occurs through inhalation and dermal contact. The principal concerns relating to exposure to concentrated forms of this material are local effects at the site of first contact and have been discussed in detail within the endpoint summary for irritation. An atmospheric concentration of 10 ppm (25 mg/m3) acetic acid is the proposed NOAEC for local irritation effects. Systemic exposure at this concentration is insignificant. For an 8 hour day spent at light work, and assuming 100% absorption of acetic acid for a worker would be 25 mg/m3 * wRV m3/kg bw = 25 * 0.144 = 3.6 mg/kg bw.

To put this intake into perspective with the known removal of acetate, it has been shown that about ~0.5 mg/kg bw acetate can be removed each minute via endogenous pathways, such as the citric acid cycle, in humans following administration of acetate in a drink (Smith et al., 2007). Daily administration of 40 mg/kg bw/day may be used as a medicinal product (Johnston & Gaas, 2006), and 25 mg/kg bw /day estimated as average human (infant) dietary intake (Ishiwata et al., 2002), with peak excursions up to 240 mg/kg bw /day (EU DAR, 2008).

In more dilute solutions, below the threshold for irritation and hence local effects, systemic availability from dermal exposure may be relevant. However for the same arguments as above, the systemic availability of acetate from dermal exposure to dilute solutions of acetic acid are insignificant.

Citations:

EFSA (2011): Scientific Opinion on the substantiation of health claims related to acetic acid and maintenance of normal blood pressure (ID 1447) pursuant to Article 13(1) of Regulation (EC) No 1924/2006; EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA); EFSA Journal 2011;9(6):2199

EFSA (2012): Scientific Opinion on the safety and efficacy of acetic acid, sodium diacetate and calcium acetate as preservatives for feed for all animal species; EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP); EFSA Journal 2012;10(2):2571

EFSA (2013): Conclusion on the peer review of the pesticide risk assessment of the active substance acetic acid; EFSA Journal 2013;11(1):3060

Ishiwater et al (2002): Daily intake of food additives in Japan in five age groups estimated by the market basket method. Eur Food Res Technol V215 pp367 -374

Johnston CS & GaasCA (2006): Vinegar: Medicinal Uses and Antiglycemic Effect. Med Gen Med. 8(2): 61:

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Although the available studies were not conducted to relevant test guidelines or specifically desigend to investigate repeated dose toxicity, they add to the weight of evidence of a lack of systemic effects via the oral route.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
This is the only study.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
This is the only study.

Local irritation/corrosion effects have been considered separately (Section 7.3).

There is sufficient evidence from animal and human investigations with acetic acid, and from human studies on acetate intake and removal to conclude that systemic toxicity is very unlikely and hence there is no justification for classification under DSD or CLP.