Naphthenic acids, zinc salts

Endpoint summary

Description of key information

There are no data on zinc naphthenate. Data from appropriate read across substances are presented, with data on naphthenic acids, zinc sulphate and zinc monoglycerolate for oral exposure.

 

Oral

Naphthenic acids

In a combined repeated dose toxicity and reproduction/developmental toxicity screening study, conducted according to OECD Test Guideline 422 and to GLP, Sprague-Dawley rats (12/sex/group) were orally administered a mixture of naphthenic acids by stomach tube (gavage, in corn oil) at doses of 0, 100, 300 or 900 mg/kg bw/day. Males were dosed for 28 days while females were dosed for a total of 39-53 days. Assessment included clinical and cage-side observations, food consumption and body weight, haematology and clinical chemistry (but no urinalysis), neurobehavioural examination, organ weights, gross pathology and histopathological examination of a wide-range of organs and tissues. A statistically significant increase in liver weight was observed in the mid- (males only) and high-dose groups; kidney weight was significantly elevated in males at the high dose. Reduced growth (not statistically significant), associated with reduced food consumption (statistically significant), was apparent in high-dose animals. Clinical observations and mortality (females only) as well as slight haemoglobin reductions and pale kidneys in males were also observed at 900 mg/kg bw/day. Additional effects on the liver, thyroid and kidney were considered either adaptive, compensatory or not relevant for humans. There were no treatment-related effects at the lowest tested dose and no neurobehavioural effects in any group. On this basis, the NOAEL for general systemic toxicity was established as 100 mg/kg bw/day, while the NOAEL for neurotoxicity was 900 mg/kg bw/day (HPVIS, 2010). [See Effects on fertility and Effects on developmental toxicity sections for further details].

 

In a limited non-guideline subchronic toxicity study, female Wistar rats (12/group) were gavaged (in water) with a mixture of naphthenic acids (isolated from Athabasca oil sands) at doses of 0, 0.6, 6 or 60 mg/kg bw/day on 5 days/week for 90 days. Relative liver, kidney and brain weights were increased in the high-dose group. Biochemical analysis revealed elevated blood amylase and hypocholesterolaemia in high-dose rats, while hepatic glycogen accumulation was also observed in five animals in this group. These results implicated the liver as a potential target organ. The study NOAEL was set at 6 mg/kg bw/day (Rogers et al., 2002).

Another non-guideline investigation involved the repeated oral [presumably gavage] administration of a mixture of naphthenic acids to male mice at 1000 mg/kg bw/day for 30 days. Observed effects included a few cases of CNS depression (without analgesia and no loss of the corneal reflex), haematological changes, weight loss leading eventually to death due to respiratory arrest, gross morphological changes in the liver and stomach, and microscopic changes in a few selected organs (Pennisi and Lynch, 1981).

Zinc

 

Matia (1981a) conducted a study to evaluate the sub chronic (13 week) toxicity of the test material in ICR mice. The study followed was equivalent or similar to OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents). Mice of both sexes were fed a diet containing test substance at 0, 300, 3,000 and 30,000 ppm for 13 weeks. The clinical signs of the animals, body weight, food, chemical and water intake, food efficiency, haematological, biochemical examination, necropsy and organ weight and histopathological examination were performed. Animals in the 30,000 ppm group showed retarded growth along with low food intake, abnormal values in a few haematological parameters, decreased water intake and significant deviations in biochemical parameters. Histopathological lesions included catarrh at the upper intestine, ulcers at the boundary of fore- and glandular stomach, proliferation of erythropoietic immature cells in the splenic red pulp as well as pancreatic lesions. Under the test conditions, NOEL of the test material in mice was determined to be 3,000 ppm (approximately equivalent to 458 mg/kg/day in male mice or 479 mg/kg/day in female mice).

 

Matia (1981b) conducted a study to evaluate the subchronic toxicity (13 week) of the test material in Wister rats. The study followed was equivalent or similar to OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents). Rats of both sexes were fed a diet containing the test material at 0, 300, 3,000 and 30,000 ppm for 13 weeks. The clinical signs of the animals, body weight, food, chemical and water intake, food efficiency, haematological, biochemical examination, necropsy and organ weight and histopathological examination were performed. Animals in the 30,000 ppm group showed retarded growth along with low food intake, abnormal values in a few haematological parameters and regressive changes of the pancreatic exocrine gland. There were no remarkable clinical signs in either sex in groups ≤ 3,000 ppm. Under the test conditions, NOEL of the test material in rats was determined to be 3000 ppm (approximately equivalent to 234 mg/kg/day in male rats and 243 mg/kg/day in female rats).

 

Edwards (1995) studies groups of 20 male and 20 female Sprague-Dawley rats which were fed zinc monoglycerolate at dietary levels of 0, 0.05 or 0.2% (equal to 0, 31.52 or 127.52 mg/kg for males and 0, 35.78 or 145.91 mg/kg bw for females, respectively) for a period of 13 weeks in a study performed according to OECD 408. A similar group was fed 1% (equal to 719 and 805 mg/kg bw/day for males and females, respectively) of zinc monoglycerolate up to day 58 of the study when a deterioration in their clinical condition (poor physical health and reduced food intake) necessitated reducing the dietary level to 0.5% (equal to 632 and 759 mg/kg bw/day for males and females, respectively). However, as no improvement occurred these rats were killed on humane grounds on day 64 of the study. These rats developed hypocupremia manifested as a hypochromic microcytic regenerative type anaemia (low haemoglobin and haematocrit, decreased MCV and MCH, and increased MCHC, red blood cell and reticulocyte count). Enlargement of the mesenteric lymph nodes and slight pitting of the surface of the kidneys were noted. Severe pancreatic degeneration and pathological changes in the spleen, kidneys, incisors, eyes and bones were observed. The testes of all males showed hypoplasia of the seminiferous tubules to a varying degree and in addition the prostate and seminal vesicles showed hypoplasia. In all but one female, the uterus was hypoplastic. All other rats survived to the end of the 13 weeks treatment. At a dietary level of 0.2% increases in plasma ALAT, alkaline phosphatase and creatine kinase were observed in males and in plasma creatine kinase in females. Total plasma cholesterol was reduced in both males and females. The changes were statistically significant but small in absolute terms. No changes in haematological parameters were seen at 0.05 and 0.2%. A dose related reduction in the quantity of abdominal fat was noted in male rats at 0.05 and 0.2%. Enlargement of the mesenteric lymph nodes was apparent in 6 out of 20 rats fed 0.2% and in one male fed 0.05%. Microscopic examination showed a reduction in the number of trabeculae in the metaphysis of the tibia of 5 male and 3 female rats fed 0.2%, 4 males and 1 female had a similar reduction in the metaphysis of the femur. Pancreatic cell necrosis was seen in both sexes at 0.2% and a slight, but statistically not significant increase could be noted at 0.05% (3 males and 1 female). This pancreatic cell necrosis was seen also in 1 control male. A reduction in the number of pigmentated macrophages in the red pulp of the spleen was observed in both sexes at 0.2% and a marginal reduction was also seen in males at 0.05%. In the animals given 0.05 and 0.2% no effects were found on the reproductive organs. Since the pancreatic cell necrosis, being without statistical significance at 0.05%, was also apparent in 1 control male and because the reduction in pigmented macrophages in the spleen was only marginal at 0.05% without any haematological changes, the dose level of 0.05% is considered as a NOAEL. This dose level is equal to 31.52 or 35.78 mg zinc monoglycerolate/kg bw for males and females, respectively, so the NOAEL in this study is 31.52 mg/kg bw (equivalent to 13.26 mg Zn2+/kg bw).

 

Conclusion for oral toxicity

Zinc naphthenate would be expected to dissociate into naphthenic acid anions and zinc cations.

 

In an OECD Test Guideline 422 combined repeated dose and reproduction/developmental toxicity screening study in rats, conducted to GLP, a mixture of naphthenic acids was administered by gavage for at least 28 days at doses of 0, 100, 300 or 900 mg/kg bw/day. Treatment-related adverse effects were observed in the mid- and high-dose animals (including increased organ weights and decreased food consumption and growth). The NOAEL for systemic toxicity was established as 100 mg/kg bw/day (HPVIS, 2010).

 

The repeated dose toxicity of water soluble zinc sulphate and zinc monoglycerolate has been examined in a total of 3 subchronic oral feeding key studies. The lowest NOAEL was determined to be 31.5 mg/kg bw/day of zinc monoglycerolate which equals a total zinc exposure of approximate 13 mg/kg bw/day (Edwards, 1995).

 

The sample of zinc naphthenate used for hazard testing (taken as representative of the range of potential compositions of zinc naphthenate) contains approximately 14.3% zinc, therefore the NOAEL value has been recalculated for zinc naphthenate. The oral NOAEL for zinc naphthenate would be 89.7 mg/kg/day.

 

Inhalation

A short-term toxicity study on zinc naphthenate via the inhalation route does not need to be conducted because reliable sub-chronic (90 days) data are available for an appropriate species, dosage, solvent and route of administration. Repeated dose oral toxicity data have been read across from naphthenic acids and zinc salts.

Furthermore, this substance is exclusively produced and used directly in grease form. The main constituent has a vapour pressure of 0.026 Pa, so the potential for the generation of inhalable forms is low. Also, the use of the grease forms will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalatory route will be unlikely to occur.

Dermal

A short-term toxicity study on zinc naphthenate via the dermal route does not need to be conducted because reliable sub-chronic (90 days) data are available for an appropriate species, dosage, solvent and route of administration. Repeated dose oral toxicity data have been read across from naphthenic acids and zinc salts.

Furthermore, zinc naphthenate is exclusively manufactured in situ in base oil and not in isolated form, so the potential for dermal contact with the isolated substance is low. Also, the use of the grease forms is expected to significantly lower the rate of adsorption through the skin, so exposure to humans via the dermal route will be unlikely to occur. The use of zinc naphthenate as grease thickeners under industrial exposure considerations precludes the relevance of the dermal route for repeated dose toxicity testing. On the assumption that the test substances will be prepared specifically for purposes for registration rather than being representative of material ‘placed on the market’, the conduct of a subacute toxicity study by the dermal route of exposure is not considered relevant for the substance.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study, key study used in EU risk assessment report for Zinc metal 2004

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

no data

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

none

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0%
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
0.05% (= male 31.52 mg/kg bw and female 35.78 mg/kg bw)
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
0.2% (= male 127.52 mg/kg bw and female 145.91mg/kg bw)
Basis:
nominal in diet

No. of animals per sex per dose:

20 males
20 females

Details on study design:

Groups of 20 male and 20 female Sprague-Dawley rats were fed zinc monoglycerolate at dietary levels of 0, 0.05 or 0.2% (equal to 0, 31.52 or 127.52 mg/kg for males and 0, 35.78 or 145.91 mg/kg bw for females, respectively) for a period of 13 weeks in a study performed according to OECD 408. Asimilar group was fed 1% (equal to 719 and 805 mg/kg bw/day for males and females, respectively) of zinc monoglycerolate up to day 58 of the study when a deterioration in their clinical condition (poor physical health and reduced food intake) necessitated reducing the dietary level to 0.5% (equal to 632 and 759 mg/kg bw/day for males and females, respectively). However, as no improvement occurred these rats were killed on humane grounds on day 64 of the study.

Observations and examinations performed and frequency:

according to guideline

Sacrifice and pathology:

according to guideline

Statistics:

according to guideline

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Ophthalmological findings:

effects observed, treatment-related

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Details on results:

The rats fed 1% of test substance for 58 days and then 0.5% to day 64 had were killed before the end of the study due to ill-health developed hypocupremia manifested as a hypochromic microcytic regenerative type anaemia (low haemoglobin and haematocrit, decreased MCV and MCH, and increased MCHC, red blood cell and reticulocyte count). Enlargement of the mesenteric lymph nodes and slight pitting of the surface of the kidneys were noted. Severe pancreatic degeneration and pathological changes in the spleen, kidneys, incisors, eyes and bones were observed. The testes of all males showed hypoplasia of the seminiferous tubules to a varying degree and in addition the prostate and seminal vesicles showed hypoplasia. In all but one female the uterus was hypoplastic.

In the main study, a dietary level of 0.2% increases in plasma ALAT, alkaline phosphatase and creatine kinase were observed in males and in plasma creatine kinase in females. Total plasma cholesterol was reduced in both males and females. The changes were statistically significant but small in absolute terms. No changes in haematological parameters were seen at 0.05 and 0.2%. A dose related reduction in the quantity of abdominal fat was noted in male rats at 0.05 and 0.2%. Enlargement of the mesenteric lymph nodes was apparent in 6 out of 20 rats fed 0.2% and in one male fed 0.05%. Microscopic examination showed a reduction in the number of trabeculae in the metaphysis of the tibia of 5 male and 3 female rats fed 0.2%, 4 males and 1 female had a similar reduction in the metaphysis of the femur. Pancreatic cell necrosis was seen in both sexes at 0.2% and a slight, but statistically not significant increase could be noted at 0.05% (3 males and 1 female). This pancreatic cell necrosis was seen also in 1 control male. A reduction in the number of pigmentated macrophages in the red pulp of the spleen was observed in both sexes at 0.2% and a marginal reduction was also seen in males at 0.05%. In the animals given 0.05 and 0.2% no effects were found on the reproductive organs.

Dose descriptor:

NOAEL

Effect level:

31.52 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

NOAEL in this study is 31.52 mg/kg bw (approximately 13.26 mg Zn2+/kg bw)

Executive summary:

Groups of 20 male and 20 female Sprague-Dawley rats were fed zinc monoglycerolate at dietary levels of 0, 0.05 or 0.2% (equal to 0, 31.52 or 127.52 mg/kg for males and 0, 35.78 or 145.91 mg/kg bw for females, respectively) for a period of 13 weeks in a study performed according to OECD 408. A similar group was fed 1% (equal to 719 and 805 mg/kg bw/day for males and females, respectively) of zinc monoglycerolate up to day 58 of the study when a deterioration in their clinical condition (poor physical health and reduced food intake) necessitated reducing the dietary level to 0.5% (equal to 632 and 759 mg/kg bw/day for males and females, respectively). However, as no improvement occurred these rats were killed on humane grounds on day 64 of the study. These rats developed hypocupremia manifested as a hypochromic microcytic regenerative type anaemia (low haemoglobin and haematocrit, decreased MCV and MCH, and increased MCHC, red blood cell and reticulocyte count). Enlargement of the mesenteric lymph nodes and slight pitting of the surface of the kidneys were noted. Severe pancreatic degeneration and pathological changes in the spleen, kidneys, incisors, eyes and bones were observed. The testes of all males showed hypoplasia of the seminiferous tubules to a varying degree and in addition the prostate and seminal vesicles showed hypoplasia. In all but one female the uterus was hypoplastic.

All other rats survived to the end of the 13 weeks treatment. At a dietary level of 0.2% increases in plasma ALAT, alkaline phosphatase and creatine kinase were observed in males and in plasma creatine kinase in females. Total plasma cholesterol was reduced in both males and females. The changes were statistically significant but small in absolute terms. No changes in haematological parameters were seen at 0.05 and 0.2%. A dose related reduction in the quantity of abdominal fat was noted in male rats at 0.05 and 0.2%. Enlargement of the mesenteric lymph nodes was apparent in 6 out of 20 rats fed 0.2% and in one male fed 0.05%. Microscopic examination showed a reduction in the number of trabeculae in the metaphysis of the tibia of 5 male and 3 female rats fed 0.2%, 4 males and 1 female had a similar reduction in the metaphysis of the femur. Pancreatic cell necrosis was seen in both sexes at 0.2% and a slight, but statistically not significant increase could be noted at 0.05% (3 males and 1 female). This pancreatic cell necrosis was seen also in 1 control male. A reduction in the number of pigmentated macrophages in the red pulp of the spleen was observed in both sexes at 0.2% and a marginal reduction was also seen in males at 0.05%. In the animals given 0.05 and 0.2% no effects were found on the reproductive organs.

Since the pancreatic cell necrosis, being without statistical significance at 0.05%, was also apparent in 1 control male and because the reduction in pigmented macrophages in the spleen was only marginal at 0.05% without any haematological changes the dose level of 0.05%, is considered as a NOAEL. This dose level is equal to 31.52 or 35.78 mg zinc monoglycerolate/kg bw for males and females, respectively, so the NOAEL in this study is 31.52 mg/kg bw (»13.26 mg Zn²⁺/kg bw)

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is performed by NTP according to GLP and valid methods, therefore it is considered to be adequate, reliable and relevant for classification. The score 1 was given bij HPVIS.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

An extension for in vivo micronucleus study was also integrated in the study

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650

Deviations:

yes

Remarks:

An extension for in vivo micronucleus study was also integrated in the study

Principles of method if other than guideline:

An extension for in vivo micronucleus study was also integrated in the study (see 7.6.2.)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

no data

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The naphthenic acids were suspended in corn oil to the appropriate concentrations and administered in 10 mL/kg doses.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Males: dosing for 28-29 days
Females: Depending on the time at which mating occurred, females were dosed for 39-53 days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations: 0, 100, 300, 900 mg/kg/day
Basis: actual ingested

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- All rats were examined twice daily for mortality and general health.
- All animals were examined approximately 1 hour after each treatment, and all unusual observations were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:Detailed physical examinations of all animals were conducted weekly.

BODY WEIGHT: Yes
- Body weights of male rats were recorded one week prior to test substance administration, on the first day of dose administration, on a weekly basis during the study and at termination. Body weights of female rats were recorded once week prior to test substance administration, on the first day of dose administration and weekly until evidence of copulation was obtained. From that point body weights of female rats were recorded on gestation days (GD) 0, 4, 7, 11, 14, 17, and 20 and on lactation days (LD) 0, 1 and 4 (termination). For females for which there was no evidence of copulation, body weights were recorded weekly until termination. Body weights of offspring were recorded on post-natal day (PND) 1 and then on PND 4, prior to termination.

FOOD CONSUMPTION:
- Food consumption determined: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No (gavage)

FOOD EFFICIENCY: no

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: No data
- On the day of scheduled termination
- The hematological investigation included measurements of total leukocyte count, erythrocyte count, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, prothrombin time, activated partial prothrombin time, reticulocyte count, mean platelet volume, red cell distribution width, hemoglobin distribution width, differential leukocyte count, and red cell morphology.
- Parameters checked in table 2 were examined.

CLINICAL CHEMISTRY: Yes
- On the day of scheduled termination
- The serum chemistry investigation included measurements of concentrations of albumin, total protein, globulin, albumin/globulin ratio, total bilirubin, urea nitrogen, creatinine, alkaline phosphatase, alanine aminotransferase, aspartate aminostransferase, gamma glutamyltransferase, glucose, total cholesterol, calcium, chloride, phosphorus, potassium, sodium, triglycerides, and bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- The potential for nervous system effects was assessed using a functional observation battery (FOB). All rats in the vehicle (corn oil) and naphthenic acid-treated groups were examined prior to dosing, after approximately 28 days of dosing, and, for females, prior to termination. The FOB procedures were based on previously developed protocols (Gad, 1982; Haggerty, 1989; Irwin, 1968; Moser et al., 1988; 1991; O’Donoghue, 1989). The testing was conducted in a sound attenuated room with a white noise generator set to operate at 70 + 10 dB. The investigators conducting the FOB were not aware of the treatment groups from which the respective animals were taken. The FOB consisted of the following: home cage observations; handling observations; open field observations; sensory observations and neuromuscular observations (Table 2). In addition there were physiological observations including body weight, body temperature and examination for catalepsy. There was also an assessment of locomotor activity which was measured electronically using a computer-controlled system with a series of infrared photobeams in a clear plastic rectangular cage. Animals were tested separately in 60 minute sessions divided into 5 minute intervals.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- At termination rats were euthanized by carbon dioxide inhalation.
- Necropsies were conducted on all animals sacrificed in extremis or at study termination.

ORGAN WEIGHTS
- Organs were removed weighed
- See table 1 and 3

HISTOPATHOLOGY: Yes
- Organs were placed in 10% neutral buffered formalin for histologic examination.
- The disposition of organs and tissues was as listed in the table 4.

Statistics:

Mean parental body weights (weekly, gestation and lactation), body weight changes and food consumption, body weight changes, absolute and relative organ weights, clinical pathology values (except for gamma glutamyltransferase), and continuous FOB values were evaluated by one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences between the vehicle control and test substance-treated groups. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Histopathological findings in the test substance-treated groups and FOB parameters yielding scalar or descriptive data were compared to the vehicle control group using Fisher’s Exact Test (Steel and Torrie, 1980). Gamma glutamyltransferase data were evaluated using the Kruskal-Wallis nonparametric ANOVA (Kruskal and Wallis, 1952) to determine intergroup differences between the vehicle control and test substance-treated groups. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunn Test (Dunn, 1964) was used to compare the test substance-treated groups to the vehicle control group.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

2 mortalities and clinical observations seen in high dose females; clinical observations in some high dose group males.

Mortality:

mortality observed, treatment-related

Description (incidence):

2 mortalities and clinical observations seen in high dose females; clinical observations in some high dose group males.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain at high dose was reduced for less than 10% in males and 4% in females versus control groups (not statistically significant).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was reduced in the high dosed male and female groups (statistically significant).

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):

Slightly reduced hemoglobin in males (statistically significant), however not in females.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Small incidental findings only, within historical range.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased liver weight in males and females of medium and high dose groups (only statistically significant at high dose). Significant increase in kidney weight in males of medium and hig dose group (only statistically significant at high dose).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Pale kidneys in the high dose males and reduction in the number of corpora lutea in the high dose females

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Hyaline-droplets in high dosed male kidneys (not relevant for humans); liver hepatocellular hypertrophy in high dosed males & females (adaptive); thyroid gland hypertrophy and vacuolation in (mid) and high dosed males & females (compensatory).

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Two high dose females were terminated on Lactation Day 2; one was sacrificed in extremis due to acute inflammation of the uterus; the other was sacrificed due to total litter loss. All other rats survived to scheduled termination. Clinical observations, which were noted only in high dose group females and approximately an hour of dosing included hunched posture; rocking, lurching, and/or swaying while ambulating; walking on tiptoes; hypoactivity; and shallow respiration. Some of the high dose group males also exhibited hunched posture.

BODY WEIGHT AND WEIGHT GAIN, FOOD CONSUMPTION
Body weight gain was reduced in high dose group males but the overall difference was less than 10% and the differences were not statistically significant. Among the females, the body weight gain in the high dose group was approximately 4% below control values but not significantly different at the end of the mating period. These differences in weight gain were associated with significantly reduced food consumption in the high dose group animals.

HAEMATOLOGY
There were some hematology changes, primarily reductions in parameters related to hemoglobin content which were considered to have been treatment related. However, as is apparent from Table 1, the differences were small and there was no consistency between males and females.

CLINICAL CHEMISTRY
The clinical chemistry values showed a similar pattern. Among males the only statistically significant differences between control were for creatinine (control value = 0.3 ± 0.1 mg/dL versus a value of 0.4 ± 0.0 in the high dose group, p < 0.01), and chloride (control value = 104 ± 1.1 mEq/L versus a value of 102 ± 1.3 in the high dose group, p < 0.01). Among the female rats, statistically significant differences were found for albumin (control = 4.3 ± 0.2 g/dL versus 4.7 ± 0.3 in the high dose group, p < 0.05), total protein (control = 6.3 ± 0.3 g/dL versus 6.7 ± 0.4 in the high dose group, p < 0.05), glucose (control = 115 ± 11 mg/dL versus 130 ± 8.0 in the high dose group, p < 0.05), cholesterol (control = 69 ± 14 mg/dL in the control versus 89 ± 19 in the high dose group, p < 0.05), calcium (control = 10.6 ± 0.4 mg/dL in the control versus 11.5 ± 0.6 in the high dose group, p < 0.01), and phosphorus (control = 3.9 ± 0.6 mg/dL versus 5.5 ± 1.2 in the high dose group, p < 0.05). All of the differences were small and within the historical range of the laboratory. Additionally, most were significant at only the 0.05 level, and there was no consistency of response between the sexes. In the absence of any corresponding pathological findings, these differences were most likely incidental.

NEUROBEHAVIOUR
There were no statistically significant differences in parameters assessed as part of the functional observation battery including home cage observations, handling parameters, open field observations, sensory observations or neuromuscular observations. There were some small differences in body weight gain as indicated previously but other physiological parameters (catalepsy, body temperature) were not affected by treatment. There were also no differences in locomotor activity patterns.

ORGAN WEIGHTS
Organ weight determinations in males revealed significant increases in weights of liver, kidney, thyroid/parathyroid and epididymis although the differences in thyroid/parathyroid and epididymal weights were only statistically different when compared on a relative to body weight basis. In females, there was a significant increase in liver weights and significant reductions in lung weights, and absolute uterine weights (table 3). The lung weights were within the historical range for the laboratory, and were not associated with any pathological changes. The uterine weights were not significantly different when compared relative to body weights. All gravid females were in lactational anaestrus and undergoing involution. Uterine weight values all fell within the historical range for the laboratory and were not associated with any gross, histopathologic or clinical pathology changes. Other than the uterine weights there were no microscopic differences in the reproductive organs of the male and female rats.

GROSS PATHOLOGY
The only notable gross observations were those of pale kidneys in the high dose males and a reduction in the number of corpora lutea in the high dose group females. Otherwise, the results of the gross examination were not remarkable.

HISTOPATHOLOGY: NON-NEOPLASTIC
The results of the pathological investigation are summarized in Table 4. Kidney changes, reported in male rats only, were consistent with hyaline-droplet nephropathy (α2u-globulin-mediated nephropathy). The liver changes, found in organs from both male and female rats from the high dose group, were described as hepatocellular hypertrophy. Other changes included cortical lymphoid depletion of the thymus in females, primarily in rats from the high dose group. Epithelial hypertrophy and cytoplasmic vacuolation of the thyroid gland was noted in all treated animals, and cytoplasmic vacuolation of the zona fasciculate in the adrenal cortex was reported in males from all treatment groups and in high dose group females. The microscopic examination also revealed minimal cardiomyopathy which occurred with increased incidence in the males in the 100, 300 and 900 mg/kg/day groups. The pathologist noted that cardiomyopathy is a common finding in rats (Greaves, 2007a), that the incidence of cardiomyopathy in the treated animals was within the historical range of the laboratory, and that the severity of cardiomyopathy in the treated male rats was similar to or less than the degree of severity found in the control animals. The pathologist also noted that the cardiomyopathy was not associated with any gross observations, organ weight changes or alterations in clinical pathology parameters.

HISTOPATHOLOGY: NEOPLASTIC
No neoplastic changes.

HISTORICAL CONTROL DATA
Not provided

OTHER FINDINGS
The gross and pathological assessments did reveal some differences that were treatment-related but were unlikely to have been toxicologically important. Liver weights were significantly increased in high dose groups of both male and female rats, and there was also a statistically significant increase in liver weight in the 300 mg/kg/day dose group in the males. The histological findings were essentially limited to minimal evidence of hepatocellular hypertrophy in the high dose group animals. As none of the liver enzyme markers were increased, this was most likely evidence of enhanced metabolic capacity and adaptive rather than adverse (Cattley and Popp, 2002). Kidney weights were significantly elevated in the male rats from the high dose group, but not in the female rats. The histological evidence revealed the presence of hyaline droplets, mostly judged to have been of minimal severity, which increased in frequency in the male rats in a dose-dependent manner. As these were not found in female rats, the histological findings and gender-specificity, suggest the kidney changes were the consequence of an α-2u-globulin-related process which is male rat specific and not relevant to humans (Hard et al., 2008; Baetcke et al., 1991; Swenberg and McKeeman, 1998).

Minimal cardiomyopathy was reported to have increased in a dose-related fashion in male rats but was not considered to have been toxicologically important. In part because this is a common observation in control rats (Greaves et al., 2007b), and, additionally because the incidence was within the historical control range of the laboratory, the severity was not greater than that seen in the control groups, and because these microscopic observations were not associated with any other gross or clinical findings.
Other changes included higher mean thyroid/parathyroid weights with corresponding epithelial hypertrophy and cytoplasmic vacuolation. The histologic changes were mostly judged as minimal. It is plausible that these changes reflected a compensatory response related to the increased metabolic capacity of the liver and more rapid turnover of thyroid hormones (Curran, 1991; Capen, 1997). Lymphoid depletion of the thymus was observed in the high dose females and microscopic findings of cytoplasmic vacuolation of the adrenal cortex were noted in the males and high dose group females. The lymphoid cortical depletion of the thymus and adrenal cortex vacuolation were considered to have been stress responses (Greaves, 2007b) although cytoplasmic vacuolation of the adrenal cortex can also occur spontaneously (Frith et al., 2000) or as the result of pharmacological effects (Greaves, 2007c). The overall no effect level for all systemic effects was 100 mg/kg/day.

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

haematology

mortality

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Remarks:

Neurotoxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

haematology

mortality

organ weights and organ / body weight ratios

Critical effects observed:

not specified

_Table 2. Results of investigations of hematology and clinical chemistry parameters which were statistically different from control values.

Parameter Measured	Corn Oil Control	100 mg/kg/day	300 mg/kg/day	900 mg/kg/day
Males, data taken at terminal sacrifice
Red Blood Cell Count (10^6/ul)^b	9.22+0.54	9.28+0.28	8.91+0.34	8.78+0.22^c
Hemoglobin (g/dL)^b	15.7+0.72	15.8+0.48	15.2+0.54	14.7+0.44^c
Hematocrit (%)^b	48.1+2.4	48.6+1.6	46.5+1.5	45.0+1.8^d
Platelet (10^3/ul)^b	854+151	885+84	803+144	976+87^d
Leukocytes, absolute (10^3/ul)^b	0.02+0.02	0.03+0.02	0.02+0.02	0.04+0.03^a
RDW (%)^b	11.4+0.4	11.5+0.4	11.6+0.4	12.5+0.6^d
HDW (g/dL)^b	2.58+0.10	2.68+0.12	2.76+0.16^c	2.77+0.27^c
Females, data taken at termination (lactation day 4)
White blood cell count^b	5.15+1.30	6.89+1.58	7.68+2.24^c	7.59+1.85^c
APTT (seconds)^b	16.8+1.9	15.9+2.3	15.8+3.1	13.9+1.4^c
Lymphocytes, absolute (10^3/ul)	3.32+0.61	4.50+1.42	5.11+1.75^c	4.96+1.60^c
Monocytes, absolute (10^3/ul)	0.11+0.10	0.24+0.21	0.21+0.12	0.35+0.23^c

1. Parameters not affected by treatment included:

a.      Males – white blood cell count, mean corpuscular volume (fL), mean corpuscular hemoglobin (pg), mean corpuscular hemoglobin content (g/dL), prothrombin time (sec), APTT (sec), reticulocytes (%), reticulocytes, absolute (10³/ul), MPV (fL), neutrophils (%), lymphocytes (%), monocytes (%), eosinophils (%), basophils (%), leucocytes(%), neutrophils, absolute (10³/ul), lymphocytes, absolute (10³/ul), monocytes, absolute (10³/ul), eosinophils, absolute (10³/ul), basophils, absolute (10³/ul).

b.      Females – red blood cell count (10⁶/ul), Hemoglobin content (g/dL), hematocrit (%), mean corpuscular volume (fL), mean corpuscular hemoglobin (pg), mean corpusc ular hemoglobin content (g/dL), platelet count (10³/ul), prothrombin time (sec), reticulocytes (%), reticulocytes, absolute (10³/ul), ), MPV (fL), neutrophils (%), lymphocytes (%), monocytes (%), eosinophils (%), basophils (%), leucocytes(%), neutrophils, absolute (10³/ul), eosinophils, absolute (10³/ul), basophils, absolute (10³/ul), Leukocytes absolute (10³/ul), RDW (%), HDW (g/dL)

Table3. Statistically significant changes in terminal body weights and organ weights (g). The data are given as mean+SD.

Parameter	Sham Control	Corn Oil Control	100 mg/kg/day	300 mg/kg/day	900 mg/kg/day
Males
Final Body Weight	467+27	454+45	448+45	439+34	412+28
Liver	15.61+1.43	13.46+2.01	13.98+2.04	15.69+1.83*	19.94+2.08^b
Kidney	3.51+0.25	3.21+0.20^a	3.38+0.39	3.53+0.33	3.77+0.46^b
Heart	1.46+0.09	1.46+0.21	1.41+0.14	1.43+0.13	1.32+0.13
Thyroid/parathyroid	0.019+0.002	0.019+0.001	0.020+0.002	0.020+0.002	0.020+0.002
Epididymis (LT)	0.57+0.14	0.60+0.05	0.60+0.04	0.66+0.05^a	0.63+0.06
Epididymis (RT)	0.62+0.04	0.62+0.06	0.61+0.03	0.66+0.04	0.65+0.06
Females
Final body Weight	335+25	313+23	301+30	294+24	289+24
Liver	13.6+2.0	11.7+1.5	12.1+1.1	13.3+1.5	17.9+2.4^b
Kidney	2.39+0.17	2.07+0.18^a	2.11+0.15	2.05+0.25	2.17+0.19
Heart	1.21+0.23	1.10+0.10	1.08+0.10	1.07+0.11	1.01+0.13
Lungs	1.36+0.13	1.40+0.13	1.26+0.12^a	1.20+0.12^b	1.20+0.07^b
Uterus/Vagina	1.07+0.19	1.00+0.14	0.86+0.08^a	0.88+0.11^a	0.85+0.12^a

a = P < 0.05, b = P < 0.01

Table 4. Summary of microscopic findings from rats following repeated treatment with refined naphthenic acids.

Sex	Males				Females
Doses,mg/kg/day	Corn Oil	100	300	900	Corn Oil	100	300	900
N	12	12	12	12	9	12	10	10
Kidney
Hyaline Droplets	0	3	10^b	11^b	0	0	0	0
Minimal	0	3	9^b	9^b	0	0	0	0
Mild	0	0	1	2
Nephropathy	0	0	2	9^b	0	0	0	0
Minimal	0	0	2	5^a	0	0	0	0
Mild	0	0	0	4
Liver
Hypertrophy, hepatocellular, centrilobular	0	0	0	8^b	0	0	0	10^b
Minimal	0	0	0	8^b	0	0	0	10^b
Vacuolation, hepatocellular	2	1	2	0	0	1	0	2
Minimal	1	1	2	0	0	1	0	2
Mild	1	0	0	0	0	0	0	0
Thymus
Depletion, lymphoid, cortex	0	0	0	0	0	1	0	5^a
Minimal	0	0	0	0	0	1	0	4
Mild	0	0	0	0	0	0	0	1
Thyroid
Hypertrophy, epithelial	0	6^a	9^b	11^b	0	3	4	8^b
Minimal	0	6^a	9^b	11^b	0	3	4	6^a
Mild	0	0	0	0	0	0	0	2
Vacuolation, cytoplasmic	0	6^a	9^b	10^b	0	3	4	8^b
Minimal	0	6^a	9^b	10^b	0	3	4	8^b
Adrenal Cortex
Vacuolation, cytoplasmic	0	2	3	2	0	0	0	2
Minimal	0	2	3	2	0	0	0	1
Mild	0	0	0	0	0	0	0	1
Heart
Cardiomyopathy
Minimal	8	5	4	4	3	3	2	4
Mild	4	7	8^a	8^a	0	0	0	0

a = P < 0.05, b = P < 0.01

Conclusions:

The overall no adverse effect level for all systemic effects was 100 mg/kg/day. The overall no adverse effect level for neurotoxicity was 900 mg/kg/day.

Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in Wistar ratswas performed by oral gavage with Naphtenic acids in corn oil. There were 3 test material treated groups (100, 300 and 900 mg/kg bw) along with a vehicle treated group (corn oil) each in 12 animals/sex/group. Male rats were dosed during premating, mating and afterwards for 28 days in total and females were dosed during premating, mating, gestation and up to day 3 post partum. In this section, only repeated dose toxicity parameters are discussed: (furher info on reproductive & developmental parameters is given in Section 7.8.1 and 7.8.2.

At the lowest dose of 100 mg/kg bw, no effects were observed both in male and female rats. At the 300 and 900 mg/kg bw doses, there were no relevant effects on clinical chemistry (only slight incidental findings) and neurobehiour at all dose levels. Further effects at the medium and high doses are described:

-  Two mortalities and clinical observations were seen in high dose females; clinical observations in some high dose group males.

-   Body weight gain at the high dose was reduced for less than 10% in males and 4% in females versus control groups (not statistically significant). This was associated with reduced food consumption in both groups (statistically significant).

-   Slightly reduced hemoglobin in males (statistically significant), however not in females.

-   Increased liver weight in males and females of medium and high dose groups (only statistically significant at high dose); significant increase in kidney weight in males of medium and high dose group (only statistically significant at high dose).

-   Pale kidneys in the high dose males and reduction in the number of corpora lutea in the high dose females.

-   Hyaline-droplets in high dosed male kidneys (not relevant for humans); liver hepatocellular hypertrophy in high dosed males & females (considered to be rather adaptive than adverse); ad thyroid gland epithelial hypertrophy and cytoplasmatic vacuolation in (mid) and high dosed males & females (considered to be compensatory related to the increased metabolic capacity of the liver and more rapid turnover of thyroid hormones).

In conclusion, NOAEL for systemic toxicity was 100 mg/kg bw, whereas NOAEL for neurotoxicity was 900 mg/kg bw, both in male and female rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

89.7 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Read across from study on zinc monoglycerolate following OECD 408

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

Additional information

There are no data on zinc naphthenate. Data from appropriate read across substances are presented, with data on naphthenic acids, zinc sulphate and zinc monoglycerolate for oral exposure. Where applicable, the results have been recalculated from the structural analogues to zinc naphthenate on the basis of the zinc content.

 

The dermal and inhalation NOAEL have not been determined as dermal and inhalation routes are not considered to be relevant routes of exposure.

Justification for classification or non-classification

There are no data on zinc naphthenate but in the human body, zinc naphthenate is expected to dissociate into zinc cations and naphthenic acid anions. Data from appropriate read across substances are presented, with data on naphthenic acids, zinc sulphate and zinc monoglycerolate for oral exposure. The NOAEL for repeated dose toxicity has been taken from subchronic data on the zinc ion and recalculated to reflect the molecular weight of zinc naphthenate.

Zinc is essential for human growth and development, neurological functions and immunocompetence. The main clinical manifestations of zinc deficiency are growth retardation, delay in sexual maturation or increased susceptibility to infections (SCF, 2003). Health specialists recommend supplementing the diet with zinc in case human diet is zinc deficient. The maximum allowable daily intake has been established to be 50 mg zinc per day. There is not sufficient experimental evidence for specific target organ toxicity based on the reversibility of the ‘adverse’ effects demonstrated. Hence, no classification is required.