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No toxicity data on adverse effects on sexual function and fertility with naphthenic acids, copper salts are available, thus the reproductive toxicity will be addressed with existing data on the individual assessment entities copper and naphthenate. Naphthenic acids, copper salts is not expected to impair fertility or show developmental toxicity, since the two moieties copper and naphthenate have not shown adverse effects in a two generation reproductive toxicity test or reproduction developmental toxicity screening test, respectively.

Endpoint conclusion:

no adverse effect observed

Endpoint conclusion:

no study available

Endpoint conclusion:

no study available

Copper

The two-generation study in the rat indicate that under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for reproductive toxicity was 1500 ppm, the highest concentration tested. The NOAEL for P1 and F1 rats and F1 and F2 offspring during lactation was 1000 ppm, based on reduced spleen weight in P1 adult females, and F1 and F2 male and female weanlings at 1500 ppm however the transient reduced spleen weights are not considered a reproductive endpoint as it did not affect growth or fertility.

In compliance with the “Definition of reproductive toxicity”, OECD document ENV/JM/MONO(2001)6 the spleen effect cannot be considered a reproductive effect as this must include:

1) Adverse effects on sexual function and fertility in adult males and females

2) Developmental toxicity in the offspring

For a compound to be considered to be a reproductive toxin “data for animal studies ideally should provide clear evidence of specific reproductive toxicity in the absence of other, systemic, toxic effects”. Therefore as the results of this study do not indicate specific reproductive toxicity at the highest dose level tested, it is proposed that copper sulphate and, after read across, copper are not classified as reproductive compounds.

A further high quality study by Chung et al (2008) provides supportive evidence. This study is a combined repeat dose and reproduction/developmental screening test, which is intended to investigate effects of repeated exposure over a relatively limited time-period and to provide initial information on effects on male and female reproductive performance. The study was conducted according to OECD test guidances (no. 422) and is considered to be of high quality. In this study, male rats were treated with copper chloride two weeks prior to mating and during the mating period (total of 30 days). Female rats were treated two weeks prior to mating, throughout mating and gestation, and until day 3 of lactation (estimated duration ~50 days). Copper chloride was administered daily as an aqueous suspension by gavage at 1.3, 5, 20 and 80 mg/kg bw/d (calculated as 0.8, 3.2, 12.9 and 51.7 mg Cu/kg bw/d). With regard to general effects of repeated exposure, systemic toxicity was evident at 80 mg/kg bw/d, consisting of some altered haematological parameters (males and females), changes in some biochemical parameters (males only) and increased incidence of femoral marrow hyperplasia (males only). With regard to reproduction/developmental effects, there were no effects on any male or female reproductive parameters investigated in this study. Effects on two developmental parameters were reported: numbers of pups with gross (icteric) lesions and numbers of runt pups, both of which were significantly increased at 80 mg/kg bw/d (51.7 mg Cu/kg bw/d).

Squamous cell hyperplasia of the stomach showed a significant, dose-related increase in males at 20 mg/kg bw/d and above and in females at 5 mg/kg bw/d and above. These effects, which were considered to be a direct effect of the irritant properties of copper chloride, are not consider relevant for the hazard profile of copper metal.

In the developmental toxicity study (Munley 2003), groups of 22 female NZW rabbits were treated orally by gavage on days 7 to 28 of pregnancy with copper hydroxide (0, 6, 9 or 18 mg Cu/kg/bw/day). A preliminary range-finding test, conducted in non-pregnant rabbits, indicated there were no marked differences between several copper compounds (including copper hydroxide, copper (I) oxide and copper oxychloride) in terms of maternal toxicity.

In the main study, maternal toxicity was evident at 9 and 18 mg Cu/kg/bw/day. Initial weight loss and reduced food intake occurred at 9 and 18 mg/kgBW/day, followed by partial recovery during the middle/late pregnancy. At the end of the study, bodyweight gain in these two groups was 31% and 72% lower than controls and total food consumption 17% and 30% lower than controls, respectively. Three deaths and two abortions occurred at 18 mg/kgBW/day which appeared to be related to treatment; necropsy of decedents and one aborted animal showed haemorrhagic and/or ulcerative changes in the stomach lining. No deaths occurred at 9 or 6 mg/kg/bw/day. At 9 mg/kg/bw/day, there were no abortions. At 6 mg/kg/bw/day, there was a single abortion on day 27. This abortion was not considered to be treatment-related in view of the absence of abortions at the higher dose level and earlier occurrence of abortions at 18 mg/kg/bw/day. At 6 mg/kg/bw/day, and bodyweight gain and food intake were only marginally lower than controls. There was no difference between treatment groups and controls in the number of pregnant females, or the number of females showing total resorption or with live offspring. There was no difference between treatment and control groups in the number of corpora lutea, implantations, embryonic deaths, live young or percentage of males in litter. At 18 mg/kg/bw/day, mean foetal weight was slightly lower than in controls (9% less). Four malformed foetuses occurred in the study: one with fused ribs (control group); one with ectopic kidney (6 mg/kg/bw/day); two with hemivertebra (18 mg/kg/bw/day). These malformations were all considered to be unrelated to treatment. With regard to fetal skeletal abnormalities, retarded ossification of pelvis and skull showed a slightly increased incidence at 18 mg/kg/bw/day and occurrence of extra ribs was increased at 9 and 18 mg/kg/bw/day compared to controls. It was noted that the occurrence of extra ribs was a common finding in all treatment groups, including the control group (64%, 67%, 80% and 87% incidence at 0, 6, 9 and 18 mg/kg/bw/d, respectively). With regard to fetal visceral abnormalities, none were recorded for any treatment or control group. In conclusion, this study demonstrated maternal toxicity (initial weight loss and reduced food intake) and effects on the fetus (increased incidence of a common skeletal abnormality) following oral exposure of rabbits to copper hydroxide at 9 mg Cu/kg/bw/day and above during pregnancy. There were no indications of fetal abnormalities associated with treatment at up to maternally toxic levels. The NOAEL for maternal toxicity and developmental effects in rabbits in this study was 6 mg Cu/kg/bw/day. Effects on the fetus were considered to be secondary to maternal toxicity and consequently not a specific effect of copper on reproduction.

Maternal toxicity, reported in this study at 9 mg/kg/bw/day, was represented by initial weight loss. These effects are considered to be local effects on the stomach in rabbits which result from gavage administration of copper hydroxide. Consequently, it is considered inappropriate to use data on maternal toxicity from this study as the basis of a repeat-dose NOAEL for copper.

The two generation oral reproduction study, performed in accordance with OECD test guideline 416, provides information on the effects of repeated exposure to the substance during all phases of the reproductive cycle including gestation. In particular, the study provides information on the reproductive parameters, and on development, growth and survival of offspring.

The NOAEL for reproductive toxicity was 1500 ppm, the highest concentration tested. The NOAELfor P1 and F1 rats and F1 and F2 offspring during lactation was 1000 ppm, based on reduced spleen weight in P1 adult females, and F1 and F2 male and female weanlings at 1500 ppm. However the transient reduced spleen weights were not considered a reproductive endpoint as it did not affect growth and fertility.

In compliance with the “Definition of reproductive toxicity”, OECD document/JM/MONO(2001)6 the spleen effect cannot be considered a reproductive effect as this must include:

· Adverse effects on sexual function and fertility in adult males and females

· Developmental toxicity in the offspring

For a compound to be considered to be a reproductive toxin “data for animal studies ideally should provide clear evidence of specific reproductive toxicity in the absence of other, systemic, toxic effects”

The dietary concentration of 1000 ppm was equivalent to mean daily intakes of copper of 15.2-23.5 mg/kg body weight/day for male rats during premating and 17.0-35.2 mg/kg body weight/day for female rats during premating, gestation and the first 2 weeks of lactation.

Although the principal aim of this study was to investigate reproduction toxicity it also provides important information on the developmental toxicity potential of the test substance. Notably, investigation of F1 and F2 litters showed no test substance related effects on the following parameters:

· pups survival, sex ratio, and survival indices during the lactation period, body weights and clinical observations during lactation,

· macroscopic examination of pups that died during the lactation period, of weanlings with external abnormalities or clinical signs and of randomly selected weanlings,

· microscopic observations of any gross findings and of liver and brain from randomly selected high-dose and control weanlings.

It is therefore considered that all major manifestations of developmental toxicity (including mortality, structural abnormality, altered growth and functional deficiency) are adequately investigated in this study.

The results of the multigeneration study should also be interpreted in conjunction with the rest of the toxicology data base for copper. The following findings are considered relevant when evaluating the reproductive and developmental toxicity potential of the test substance:

· Subchronic and chronic studies show no adverse effects on reproductive organs or endocrine functions,

· Copper salts show no indication of genotoxicity,

It is also important to consider that copper is an essential element and many countries recommend an increased dietary intake of copper during pregnancy. This increased recommendation is because a foetus requires copper levels up to 10 times adult levels. The copper is absorbed across the placenta and is required for healthy growth and development, especially in blood maturation, bone development, heart development and function, brain development and function and the function of 20 key enzymes (Ralph & McArdle, 2001).

The existing toxicology data package therefore supports the conclusion that copper has no reproductive or developmental toxicity potential.

Naphthenate

A key combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in Wistar rats was performed by oral gavage with Naphthenic acids in corn oil (HPVIS, 2010). 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 reproductive toxicity parameters are discussed; further info on repeated & developmental parameters is given in Section 7.5.1 and 7.8.2. Target organ findings were identified at the dose of 900 mg/kg bw, whereas 100 mg/kg bw was considered as the NOAEL for systemic toxicity.

No reproductive effects were identified up to 900 mg/kg bw . There were no weight differences in any of the other organs nor any pathological changes in the reproductive organs up to the highest dose tested (900 mg/kg/day). The NOAEL for mating and reproductive organ effects was 900 mg/kg/day.

There was also a reproductive toxicity study in male New Zealand White rabbits dermally exposed to 2 mL undiluted Calcium naphthenate for 6 hours daily for 5 days each week over 10 weeks (Dix and Cassidy, 1983). Half of the males of each group were killed and necropsied after mating. The remaining males were weighed weekly and necropsied approximately 12 weeks later. Macroscopic and microscopic examinations of the male reproductive tracts were carried out on all rabbits. The females were necropsied on day 29 of gestation. Numbers of corpora lutea, total implantations, pre-and post-implantation losses and numbers of viable foetuses were recorded. Calcium naphthenate in carrier oil did not show any effect on the reproduction when applied dermally on male rabbits. Since the naphthenate ion is considered as the toxic entity of the whole molecule read across of these findings to naphthenic acid is justified. Although no effects were observed, supporting the main study, the data were limited and the study was disregarded for risk assessment.

A supporting combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in Wistar rats was performed by oral gavage with Naphthenic acids in corn oil (HPVIS, 2010). 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 developmental toxicity parameters are discussed: (further info on repeated & reproductive parameters is given in Section 7.5.1 and 7.8.1). Target organ findings were identified at the dose of 300 and 900 mg/kg bw, whereas 100 mg/kg bw was considered as the NOAEL for systemic toxicity. Treatment of Sprague-Dawley rats with refined naphthenic acids had no apparent effects on mating and did not produce malformations at the highest dose tested (900 mg/kg/day). There were significant reductions in number of offspring, number live born and offspring body weights at 300 and 900 mg/kg, which were considered secondary findings at the maternally toxicity dose levels. The overall no observed adverse effect level was 100 mg/kg/day.

A disregarded study was available in pregnant female Wistar rats dosed at 6 and 60 mg/kg bw with Naphthenic acids obtained from Athabasca oil sands (AOS) from 14 days prior to pairing and continued throughout the mating and gestational periods until study termination on post-natal day 3 (Rogers, 2003). Reproductive toxicity testing demonstrated dramatic effects on female fertility at an oral dosage of 60 mg/kg/day during pre-breeding, breeding and gestation. While control and low dose (6 mg/kg/day) animals achieved 93 (13/14) and 100% (13/13) reproductive success, respectively, only 7% (1/14) of females dosed at 60 mg/kg/d successfully bore a litter. Total cholesterol of the latter group was 30% lower than controls. Mating and ovulation were comparable amongst control and dose groups, while fetal malformations were not apparent in any offspring. Results suggest that the dose-related infertility may be associated with poor embryonic implantation, an effect that might be secondary to depressed sex hormone production requiring cholesterol as a precursor.

Available data suggest values that warrant no classification for reproductive and developmental toxicity under Regulation 1272/2008 (CLP).

Naphthenic acids, copper salts

Naphthenic acids, copper salts is not expected to impair fertility, since the two assessment entities copper and naphthenic acid have not shown toxicity to the reproduction in a range of test systems. For further information on the toxicity of the individual moieties, please refer to the relevant sections in the IUCLID and CSR. Information on the individual moieties copper and naphthenic acid will be used for the hazard assessment and, when applicable, for the risk characterisation of naphthenic acids, copper salts. For the purpose of hazard assessment of naphthenic acids, copper salts, the point of departure for the most sensitive endpoint of each moiety will be used for the DNEL derivation. In case of naphthenic acid in naphthenic acids, copper salts, the NOAEL of 100 mg/kg bw/day for the reproductive toxicity will be used. In case of copper the Tolerable Upper Intake Level (UL) of 5 mg/kg bw/day based on human data will be used.