Malic acid

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Experimental data published in peer reviewed journal

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

2 rather than 3 treatment groups investigated

GLP compliance:

no

Remarks:

Study pre-dates implementation of GLP regulations

Limit test:

no

Species:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: No data
- Age at study initiation: (P) x approximately 3 wks; (F1) x 14 wks (at mating)
- Weight at study initiation: (P) Males: no data; Females: no data; (F1) Males: no data; Females: no data
- Fasting period before study: No data, assumed not applicable
- Housing: No data
- Diet (e.g. ad libitum): No data, assumed ad libitum
- Water (e.g. ad libitum): No data, assumed ad libiutum
- 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

Route of administration:

oral: feed

Vehicle:

not specified

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

Details on mating procedure:

- M/F ratio per cage: No data
- Length of cohabitation: No data
- Proof of pregnancy: No data, referred to as day ? of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. No data
- Further matings after two unsuccessful attempts: No data
- After successful mating each pregnant female was caged: No data

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

From 9 weeks prior to mating of P1 animals through to weaning of 2nd generation animals

Frequency of treatment:

Continuous (in feed)

Details on study schedule:

- F1 parental animals not mated until approximately 100 days after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 14 weeks

Remarks:

Doses / Concentrations:
0, 1000 and 10000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

10 males / 20 females / group

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: No data
- Rationale for animal assignment (if not random): No data

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: No data

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

No data

Litter observations:

No data
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes but no details
- If yes, maximum of 8 pups/litter; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS: No data

Postmortem examinations (parental animals):

No data

Postmortem examinations (offspring):

No data
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
No data

Statistics:

No data

Reproductive indices:

No details available

Offspring viability indices:

No details available

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain of males slightly reduced prior to mating

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Body weight gain of males slightly reduced prior to mating

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Body weight gains of males of the P1 generation groups treated with malic acid were slightly decreased compared to control animals prior to mating. Body weights in females were comparable (with controls).
Feed consumption was similar for test and control animals.
Survival was similar for test and control animals.
Appearance and behaviour were similar for treated and control rats of the P1 generation.
None of the P1 animals died during the FIA or F1B phase.

Dose descriptor:

NOAEL

Effect level:

10 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Laboured respiration / wheezing during weaningk

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

For all litters, the various indices, litter sizes, and pup body weights were comparable among test and control animals.
Necropsied pups from three of the low-dose F1A litters had rough surfaces on the spleen.
The number of pups that were weak or had laboured respiration during lactation was increased in the high-dose group F2A litters. No abnormal findings were reported at necropsy.

The P2 test and control animals were similar throughout the study. Wheezing was observed in all groups during the F2B phase.
At necropsy of the F2A litters, renal discoloration was noted in two animals, dark renal medullas were noted in four animals, rough surfaces on the spleen were noted in four animals and white foci on the spleen were seen in three weanlings of the low-dose group. Renal discoloration (three animals), dark red corticomedullary zones (three animals), dark renal medullas (three animals), rough surfaces on the spleen (two animals), and a firm, enlarged, irregularly-shaped caecum with a hole penetrating it (one animal) were observed in high-dose weanling animals.
In the F2B litters, weakness and laboured respiration were observed in a few low-dose pups, and the renal pelvis of one high-dose pup was dilated at necropsy. The animals of the F2B generation delivered by caesarean section exhibited no meaningful differences between test and control animals in the number and placement of implantation and resorption sites or in the number, weight, or length of live neonates, and none of the neonates died.
The skeletal development of the F2B neonates was similar between test and control animals.
Those slight differences in developmental indices that were observed were considered to be within the range of normal variations in foetal development.

Dose descriptor:

LOAEL

Generation:

F2

Effect level:

10 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical signs

Reproductive effects observed:

not specified

Conclusions:

Administration of malic acid over 2 generations had no clear effect on fertility or development. The LOAEL from the study may be regarded as 10000 ppm.

Executive summary:

Reproductive toxicity has been investigated with a 2-generation study using methods similar to those described in OECD TG 416. Administration of malic acid over 2 generations had no clear effect on fertility or development. The LOAEL from the study may be regarded as 10000 ppm.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LOAEL

520 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproductive toxicity has been investigated with a 2 -generation study in the rat using methods similar to those described in OECD TG 416. Administration of malic acid over 2 generations had no clear effect on fertility or development. The LOAEL from the study may be regarded as 10000 ppm. The publication "Evaluation of the Health Aspects of Malic Acid as a Food Ingredient" prepared for the US FDA in 1975 estimates this LOAEL be equivalent to approximately 40 mg/kg/day. However, conversion factors described by EFSA (Guidance on selected default values to be used by the EFSA Scientific Committee, Scientific Panels and Units in the absence of actual measured data, EFSA Journal 2012; 10(3): 2579) suggest a LOAEL of 520 mg/kg/day to be more realistic.

Effects on developmental toxicity

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Experimental data published in peer reviewed journal

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

no

Remarks:

Study pre-dates implementation of GLP regulations

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

No data

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: No data

VEHICLE
- Concentration in vehicle: Up to 175 mg/mL
- Amount of vehicle (if gavage): Up to 2 mL/kg

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: No data
- Length of cohabitation: No data
- Proof of pregnancy: vaginal plug, referred to as day 0 of pregnancy

Duration of treatment / exposure:

Days 6-15 of gestation

Frequency of treatment:

Daily

Duration of test:

20 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

3.5 mg/kg bw/day (nominal)

Dose / conc.:

16.2 mg/kg bw/day (nominal)

Dose / conc.:

75.4 mg/kg bw/day (nominal)

Dose / conc.:

350 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25-29 / group
Number gravid/number mated: 0.0 mg/kg (controls) = 23/25; 3.5 mg/kg (low dose) = 20/25; 16.2 mg/kg = 21/29; 75.4 mg/kg = 22/25; 350 mg/kg (high dose) = 26/28

Control animals:

yes, concurrent vehicle

Details on study design:

No data

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations included: Appearance, behaviour and food consumption.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 6, 11, 15 and 20 of gestation

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Uterus and urogenital tract

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes, 1/3 of litter
- Skeletal examinations: Yes, 2/3 of litter
- Head examinations: No data

Statistics:

No data

Indices:

No data

Historical control data:

No data

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:no effects

Dose descriptor:

NOAEL

Effect level:

350 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: lack of observed maternal toxicity

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEL

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Lack of treatment-related effects

Abnormalities:

no effects observed

Developmental effects observed:

no

Summary of findings of the embryo-foetal toxicity study in rats.

Daily Dose (mg/kg)	0 (Control)	3.5	16.2	75.4	350.0
Dams/Does:
No. Used/No. Pregnant	25/23	25/20	29/21	25/22	28/26
No. Died or Sacrificed Moribund	0	0	0	0	2
No. Aborted or with Total Resorption of Litter	0	0	0	0	0
Clinical Observations	-	-	-	-	-
Necropsy Observations	-	-	-	-	-
Body Weight (g^a)	346	348	339	325	321
Total No. dams with resorptions	2	0	3	5	2
Mean % resorptions	8.70	0.00	14.3	13.6	8.33
Mean No. Implantations	11.4	12.0	11.3	10.4	10.3
Litters:
No. Litters Evaluated	23	20	21	22	24
Mean No. Live Foetuses	11.3	12.0	11.2	10.1	10.3
No. of Litters with dead Foetuses	2	2	2	2	1
Mean Foetal Body Weight (g)	3.80	3.93	3.79	3.79	3.78
Foetal Sex Ratio (♂/♀)	0.77	0.99	0.88	1.18	0.89
Foetal Anomalies:
Gross External	0	0	0	0	0
Visceral Anomalies	0	0	0	0	0
Skeletal Variations	232	151	105	171	138
Key: - = No noteworthy findings       a = At termination

Conclusions:

Administration of malic acid to pregnant rats for 10 consecutive days during the critical period of organogenesis had no clear effect on maternal or foetal survival. The number and nature of abnormalities of the soft or skeletal tissues were no different from those seen in control animals.

Executive summary:

Developmental toxicity has been investigated using methods similar or equivalent to those described in OECD TG 414. Administration of malic acid to pregnant rats for 10 consecutive days during the critical period of organogenesis had no clear effect on maternal or foetal survival. The number and nature of abnormalities of the soft or skeletal tissues were no different from those seen in control animals. The NOAEL from the study was 350 mg/kg body weight/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

350 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental toxicity has been investigated in rats, mice and rabbits using methods similar or equivalent to those described in OECD TG 414. Administration of malic acid to pregnant animals during the critical period of organogenesis had no clear effect on maternal or foetal survival. The number and nature of abnormalities of the soft or skeletal tissues were no different from those seen in control animals. The NOELs from the studies were: Rats - 350 mg/kg body weight/day; Mice - 266 mg/kg body weight/day; Rabbits - 300 mg/kg body weight/day.

Justification for classification or non-classification

Based on the available information, malic acid does not require classification or labelling according to Directive 677548/EEC (DSD) and to Regulation 1272/2008/EC (CLP).