Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Lack of fertility effects observed in reproductive toxicity studies with soluble Ni compounds (Ambrose et al., 1976; Smith et al., 1993; RTI, 1988a,b; NTP, 1996; SLI, 2000a,b) combined with toxicokinetic data (Ishimatsu et al., 1995) provide sufficient justification that nickel metal should not be considered a reproductive toxicant with regards to fertility effects.

Additional information

Data requirements for nickel metal are fulfilled by reading across the negative results from fertility studies with soluble nickel compounds.

There are no existing reliable animal studies examining fertility endpoints with micron powder and massive nickel metal that fulfill the Annex X endpoint requirements for fertility. However, potential fertility impairment due to oral or inhalation exposure to nickel compounds (including the most bioavailable forms) has been extensively studied in reliable studies, and no effects on fertility have been found. There are several reliable 13 week and one- and two-generation studies utilizing inhalation or oral administration of water-soluble nickel compounds in rats that have not indicated adverse effects on fertility, estrous cycling, sperm parameters, vaginal cytology, copulation and fertility indices, precoital intervals, gestation lengths, gross necropsy findings and histopathology (Ambrose et al., 1976; Smith et al., 1993; RTI, 1988a,b; NTP, 1996; SLI, 2000a,b). While some effects have been reported in studies in mice with soluble nickel (e.g.Pandey and Srivastava, 2000), the EC reviewed this data and did not consider them sufficient to classify soluble nickel compounds for fertility effects (EURAR, 2008-2009a,b). Because the reproductive toxicity effects are related to the bioavailable nickel ion, the lack of fertility effects following exposure to water-soluble nickel compounds (which have higher bioavailability) is relevant to water-insoluble nickel compounds and nickel metal, which have lower bioavailability (Ishimatsu et al., 1995). Since adverse fertility effects are not observed with the most-bioavailable forms of nickel, they will not be observed with less-bioavailable forms. Therefore, while water-soluble nickel compounds are classified as Repro Cat 1B due to developmental effects in rats, neither water-soluble nickel compounds, nickel metal, nor insoluble nickel compounds carry harmonized classifications for fertility effects (see Part 3 of Annex VI; CLP Regulation, 2009).

Effects on developmental toxicity

Description of key information

It had been considered that data from reproductive toxicity studies with Ni sulphate hexahydrate (SLI, 2000) and Ni chloride hexahydrate(RTI, 1988) combined with inhalation and oral toxicokinetic data (Oller et al., 2008; Ishimatsu et al., 1995) and oral toxicity data for nickel metal (FDRL, 1983) provide sufficient justification that nickel metal is poorly absorbed and thus was not considered to be a developmental toxicant. New information on the potential developmental toxicity (i.e. perinatal mortality) effects of Ni metal micron powder (Kong et al., 2014) has called into question the robustness of the previous weight of evidence evaluation of Ni metal’s developmental toxicity. Therefore, an EOGRTS is proposed (see attached proposal) to clarify the hazard identification and classification for this endpoint. It is important to note that current DNELs and RMMs based on respiratory tract inflammation and cancer effects are sufficient to protect workers from developmental effects while the testing is being carried out.

Additional information

No higher-tier reliable animal studies exist examining the developmental effects of micron powder and massive forms of nickel metal. There are several reliable studies of the developmental effects of water-soluble nickel compounds in rats and mice and these studies have demonstrated adverse developmental effects. However, a high-quality epidemiological study of female workers exposed to nickel metal and nickel compounds exists (Vaktskjold et al.2006, 2007, 2008a,b) that have not indicated adverse developmental effects in humans.

In 2004, the European Commission (EC) reviewed the available animal and human data on the developmental toxicity of nickel substances and concluded that there was enough evidence from animal studies to assign a harmonized classification of animal reproductive toxicant (Cat 1B) developmental effects to the readily water-soluble nickel compounds such as nickel sulfate hexahydrate (see Part 3 of Annex VI; CLP Regulation, 2009; EURAR, 2008-2009b,c). This classification applied specifically for developmental effects (i.e.,perinatal mortality) observed in the rat studies available at that time (Ambroseet al., 1976; RTI, 1988a,b; Smithet al., 1993; SLI 2000a,b). Animal studies demonstrated that absorption of nickel ion into systemic blood circulation after high oral exposure (e.g. LOEAL of 2.2 mg Ni/kg bw/d) to water-soluble nickel compounds during pregnancy increased the death rate of the offspring (Ambrose et al., 1976; RTI, 1988a,b; Smithet al., 1993; SLI, 2000a,b). While no other developmental effects, including malformations (i.e.,teratogenesis), were identified in a rat PNDT study with water-soluble nickel chloride hexahydrate at the maximum tolerated dose of 42 mg Ni /kg bw/day (RTI, 1988a,b), nickel chloride was shown to cause malformations (e.g.microphthalmia) in a PNDT study in mice at 46 mg/kg bw/day and other teratogenic effects at higher doses (Saini et al., 2013). 

A key factor in the hazard assessment (and classification) of nickel and nickel compounds is the bioavailability of the nickel ion released from the different nickel compounds as well as metallic nickel. The EC determined that less-soluble nickel compounds, such as nickel oxides, sulfides, and metallic nickel, did not meet the criteria to be classified as reproductive toxicants (EURAR, 2008-2009a,b). This was based on toxicokinetic data indicating the much-reduced oral bioavailability of Ni ion from these substances compared to soluble nickel compound exposure in rats (Ishimatsu et al., 1995). In the case of nickel metal, 100-fold lower oral absorption was observed compared to nickel chloride or nickel sulphate.

The results of animal studies with highly bioavailable soluble nickel compounds indicates that soluble nickel compounds can produce malformations in rodents in a dose-dependent manner with an identifiable threshold for effects. While no teratogenic effects were observed in a PNDT study with nickel chloride with rats at levels up to 42 mg Ni/kg/day (RTI, 1988), malformations were observed in a PNDT study with nickel chloride in mice at exposure levels of 46 mg Ni/kg/day and higher (microphthalmia at 46 mg Ni/kg/day and other malformations at levels ≥92 mg Ni/kg/day, which also caused maternal toxicity) (Saini et al., 2013). An exposure level of 42 mg water soluble Ni/kg/day is ~10,000-fold higher than normal dietary nickel (which is less bioavailable). 

Toxicokinetic data indicates that oral exposure to metallic nickel (powder or massive form) would not allow enough absorption of nickel ions from metallic nickel to exceed the threshold for teratogenic effects reported in the study in mice with nickel chloride (i.e. one study reported 100-fold lower oral absorption of nickel from nickel metal compared to soluble nickel compounds in rats; Ishimatsu et al., 1995). Lower absorption of Ni ions from nickel metal compared to soluble nickel compounds is consistent with the very high LD50 and LC50 values for nickel metal (FDRL, 1983, 1985 respectively), demonstrating low acute oral and inhalation toxicity. Even if the difference in absorption were only 10-fold, a daily dose of 460 mg/kg of metallic nickel would be needed to deliver the same systemic dose as the 46 mg Ni/kg study with soluble nickel. This would require exposure levels ~100,000-fold higher than normal dietary intake levels. 

Epidemiological studies have not shown developmental effects such as malformations to be associated with workplace nickel exposures (Vaktskjold et al.,2006, 2007, 2008a,b) This work is important in demonstrating that no hazard for reproductive impairment from nickel compound and metal exposure exists under the conditions of the study, which had extremely high exposure levels which are no longer present in current refinery operations. Thus, the teratogenic effects observed in rodents 1) may not be relevant to humans or 2) may not have been observed in the exposed human population because even the highest achievable female workers’ exposure (e.g.~179 µg Ni/l in urine) is lower than those achieved in rats at the LOAEL (2.2 mg soluble Ni/kg bw) for developmental effects (2300 µg Ni/L in urine). Furthermore, the developmental LOAEL in rats is 40-fold lower than the LOAEL for teratogenic effects in mice (46 mg soluble Ni/kg bw) consistent with the lack of effects among refinery workers. In either case, for classification and risk assessment purposes, the relevance of the positive teratogenic results in mice with soluble nickel compounds (at what seem to be unachievable human exposures) need to be considered together with the negative results in human studies at Monchegorsk (for the highest exposed human population) in a weight of evidence approach (Haberet al., 2017). At the very least, the data indicates that humans do not appear to be greatly more sensitive to developmental toxicity effects (including teratogenicity) of nickel ion than rats. While the exact mode of action for the microphthalmia induced by soluble Ni in mice is not known, the existing data demonstrates that these are clearly threshold-mediated effects. Because the human study correlated the systemically available nickel levels to reproductive outcomes, the lack of teratogenicity effects is also relevant for exposures to nickel compounds and nickel metal.

While high exposures of soluble nickel chloride (e.g., 46 mg Ni/kg) have been associated with malformations in rodents in a dose-dependent manner with an identifiable threshold for effects, toxicokinetic data indicates that nickel metal has a much lower absorption (i.e., 100-fold lower) and thus much lower bioavailability. This indicates that oral exposure to metallic nickel (powder or massive form) would not allow enough absorption of nickel ions from metallic nickel to exceed the threshold for teratogenic effects reported in the study in mice with nickel chloride. Lower absorption of Ni ions from nickel metal compared to soluble nickel compounds is consistent with the very high LD50 and LC50 values for nickel metal (FDRL, 1983, 1985 respectively), demonstrating low acute oral and inhalation toxicity. Furthermore, epidemiological studies have not shown developmental effects such as malformations to be associated with workplace nickel exposures including nickel metal (Vaktskjold et al.,2006, 2007, 2008a,b) and in vivo micronucleus assays have not shown exposure to soluble nickel to cause effects on rapidly dividing tissues. When all the data are considered in a weight of evidence approach, the data do not support nickel metal (powder or massive form) causing teratogenic effects. This is in agreement with the EC determination that less-soluble nickel compounds, such as nickel oxides, sulfides, and metallic nickel, did not meet the criteria to be classified as reproductive toxicants (EURAR, 2008-2009a,b).

Effects through Lactation

While no higher-tier reliable studies specifically examining the effects of nickel exposure through mother’s milk were found, the existing two-generation studies with soluble nickel compounds provide no evidence of nickel-specific effects through lactation (Ambrose et al., 1974; RTI, 1988a,b; Smith et al., 1993; SLI, 2000a,b). In these studies, no effects were observed through the period of lactation, including test article-related clinical signs, weight gain, viability, and gross necropsy results in pups, utilizing oral administration of water-soluble nickel compounds (i.e.,the most bioavailable forms) to the mothers. These findings are also relevant for insoluble nickel compounds and nickel metal. While water-soluble nickel compounds are classified as Repro Cat 1B due to developmental effects in animals as reviewed above, they do not carry the requirement for labeling for effects through lactation (see Part 3 of Annex VI; CLP Regulation, 2009) and this is consistent with existing data.

Fulfilling data Requirements for Nickel Metal: 2010 to 2017

The reproductive toxicity data requirements are described under REACH Annex X, 8.7 (and column 2 thereof). Until the present update, adaptations have been used to fulfil the reproductive toxicity (fertility and developmental toxicity) data requirements for nickel metal by (1) reading across the lack of fertility effects from the studies conducted with soluble nickel compounds (i.e.,most bioavailable) to nickel metal (i.e.,less bioavailable) and (2) utilizing the rat data on developmental effects of soluble nickel compounds combined with bioavailability and toxicokinetic data on nickel metal to indicate, in a weight of evidence approach, that no developmental effects would be expected after exposure to nickel metal. Furthermore, the lack of malformations in rat (i.e.PNDT) and human studies exposed to soluble nickel compounds were read across to nickel metal to adapt the requirements for two PNDT studies (see Appendix B5 of the April 2017 version of the nickel metal CSR from more information on the previously-employed read-across strategy for reproductive toxicity endpoints).

A key factor in the hazard assessment of nickel metal and nickel compounds for reproductive toxicity is the bioavailability of the nickel ion released from the substances following exposure. While perinatal effects were observed after exposure to soluble Ni compounds, the available toxicokinetic data showed that oral absorption of nickel from insoluble nickel compounds and metallic nickel in rats is many-fold lower than that of soluble nickel compounds (Ishimatsu et al., 1995). In the Ishimatsu et al.study, a 100-fold lower oral absorption of nickel from nickel metal (micron-size powder) than from soluble compounds was observed. Thus, oral exposure to the registered nickel metal powder was not expected to allow enough absorption of nickel to exceed the threshold for developmental effects. Likewise, animal toxicokinetic data predicted that repeated inhalation exposure to metallic nickel (MMAD ~ 2mm) in rats would not increase nickel blood levels above the threshold for nickel-mediated reproductive toxicity in rats (Oller et al., 2008).[2]

These observations were further supported by the fact that micron-size nickel metal is not classified for acute toxicity by oral or inhalation routes in the 1st ATP to the CLP Regulation (see Part 3 of Annex VI; CLP Regulation, 2009) based on existing studies (FDRL 1983, 1985). The low acute toxicity of nickel metal indicates expected low bioavailability by all routes of exposure.[3] In addition, 28-day and 13-week inhalation studies with nickel metal did not reveal adverse macroscopic (28-day) or macroscopic and histopathologic (13-week) observations in ovaries, oviducts, uterus, mammary glands (females only), epididymides, testes, prostate, or seminal vesicles in rats (WIL Research Laboratories, 2002; 2004). Further, there were no effects on organ weight for epididymides (only reported in 28-day study), ovaries with oviducts, or testes (WIL Research Laboratories, 2002; 2004). Further, a 2-year inhalation study reported a 15% decrease in absolute testes weight at the maximum-tolerated dose of 0.4 mg/m3, but no testicular atrophy, degeneration of seminiferous tubules, tubular dilation, necrosis, inflammation, or hyperplasia were seen during histopathologic evaluation, nor were any weight or histopathological changes seen in the ovaries or uteri/cervixes of the female rats at any exposure level (WIL Research Laboratories, 2008). Finally, the negative results of the well-conducted epidemiological study in workers (exposed to various chemical forms of nickel including nickel metal) described above provided additional support for adapting and waiving the need to have an EOGRTS with nickel metal.

The results (lack of malformations) of a PNDT rat study with a soluble nickel compound (i.e.,highest bioavailability) (RTI, 1988a,b) was read-across to metallic nickel to fulfil the reproductive toxicity data requirements (first PNDT) for teratogenicity effects under REACH. In this study, no evidence of teratogenic effects was reported in a multi-generational study where the F2b generation was subjected to a classical teratology assessment (RTI, 1988a,b). The F2b generation was delivered by Caesarean section on gestation day 20 and were examined for external, internal, and skeletal malformations mimicking the protocol of a stand-alone PNDT study, but with extended exposure of the parental animals. This F2b cohort showed no exposure-related adverse effects. The lack of adverse effects in the F2b generation, examined on gestation day 20, indicated that the developmental effects in the other generational cohorts (i.e., perinatal mortality) were expressed during the perinatal or postnatal periods and not during gestation (RTI, 1988a,b).

Finally, rat micronucleus studies in bone marrow after repeated oral exposure to a soluble nickel compound did not show adverse effects, even though nickel levels in plasma and bone marrow were increased >30 times and 4 times, respectively, over controls (Oller and Erexson, 2007). This study confirmed that rapidly dividing tissues (such as those in the developing organism) are not preferentially affected by nickel exposure.

A requirement for a second PNDT was waived for all nickel compounds and nickel metal based on the lack of a correlation between nickel exposure and observed (sex organ and skeletal) malformations in the human reproductive studies of nickel-exposed workers reviewed above (Vaktskjold et al.,2006, 2007; 2008a,b). Specific endpoints, their assessment methods, and the number of subjects included were:

1.                  genital malformations in newborns of female nickel-refinery workers were examined with a register-based, nested case-control study (n= 103 cases; 23,038 controls);

2.                  small-for-gestational-age newborns of female refinery workers exposed to nickel were examined with a register-based, nested case-control study (n= 2,096 cases; 20,740 controls);

3.                  spontaneous abortions among nickel-exposed female refinery workers were examined with a case-control study (n=184 cases, 1,691 controls);

4.                  maternal nickel exposure and congenital musculoskeletal defects were examined with register-based, nested case-control study (n=341 cases, 22,624 controls). 

In all studies, nickel exposure was not associated with adverse pregnancy outcome for any of the endpoints examined. Such large studies provide adequate statistical power to support a conclusion to exclude the risk of reproductive effects (e.g. malformations) from human exposure to the chemical (EMA/CHMP Guideline, 2006). 

The weight of evidence from all data discussed above demonstrated lowin vivobioavailability by oral, inhalation and dermal routes, and was consistent with the lack of classification for reproductive toxicity of nickel metal, and was viewed as sufficient to adapt the data requirements for the reproductive toxicity endpoint.

Fulfilling data Requirements for Nickel Metal: 2018

We have now become aware a mouse PNDT study with nickel chloride. Saini et al. (2013) studied the effects of oral (gavage) exposure during gestation (GD 6-13) of Swiss albino mice to Ni chloride hexahydrate at doses of 0, 46, 92 and 185 mg Ni/kg bw/d. Maternal toxicity (decrease feed consumption, water intake and bw) was observed at doses ≥ 92 mg Ni/kg bw/d and fetotoxicity (decreases in bw), embryotoxicity (decrease in the number of live fetuses/dam, increases in post-implantations losses and resorptions at high dose), and teratogenicity (malformations such as open eyelids, club foot, umbilical hernia, ophthalmic anomalies, hydrocephaly, reduced ossification, dose-dependent increase in skeletal anomalies) were observed at doses ≥ 92 mg/kg bw/d (microphthalmia already at 46 mg/kg bw/d). The NOAEL for maternal toxicity was 46 mg Ni/kg bw/d and the LOAEL for developmental toxicity was 46 mg Ni/kg bw/d. These doses are much higher than the NOAEL of 1.1 Ni/kg bw/d and LOEAL of 2.2 mg Ni /kg bw/d for perinatal mortality effects in the rat 2-gen study with nickel sulphate. While the perinatal mortality effect in rats will still drive the risk characterization of nickel compounds, the mouse study is included in the weight of evidence analysis used to fulfill the requirement for a second-species PNDT (in addition to the human reproductive studies of nickel-exposed workers reviewed above).

We have also become aware of a study reporting the effects of nano-size metallic nickel particles on rat reproduction (Kong et al., 2014). Study results indicated that oral exposure to nickel metal nanoparticles at relatively high levels (5-45 mg/kg bw) was able to replicate the kind of effects that have been seen previously with water-soluble nickel compounds. This is likely due to the fact that nano-size particles, having a much larger surface area than micron and massive forms of nickel, release higher nickel ion levels in biological fluids. This could lead to significant absorption of nickel ions from nano-nickel which can lead to high Ni ion bioavailability. While there are uncertainties associated with the study, such as the product code of the test material listed in the publication matching a tungsten rather than a nickel nano powder, and that the nano nickel metal test article was sonicated prior to administration, potentially artificially increasing the bioavailability of nickel ions, this study ostensibly demonstrated reproductive toxicity (perinatal mortality) from oral exposure to a suspension of nickel metal nano particles.[4]

While the Kong et al.study included three exposure levels of nickel nanoparticles, and thus was able to examine the dose-response for the nano-size powder, the study also included one group of animals exposed to micron-size particles of nickel metal, and this was at a very high level (45 mg/kg). The animals in this group also experienced some degree of reproductive toxicity (perinatal mortality), although it was significantly lower than for the equivalent mass dose of nano powder. This result was surprising, in that micron-size nickel metal powder was not expected to be sufficiently bioavailable to cause positive effects even at this very high dose of 45 mg/kg. A difference in potency between nano and micron would be expected, as there would be lower oral absorption of nickel from micron-size metal powders than from nano-metal powders or soluble nickel compounds. However, the difference in the potency to produce reproductive effects between nano- and micron-size nickel metal particles observed in the Kong et al. (2014) study (e.g. about 6-fold) is smaller than the previously reported 100-fold difference in absorption between nickel metal and soluble nickel compounds based on toxicokinetic studies (Ishimatsu et al., 1995). 

Unfortunately, a proper dose-response for metallic nickel micron-size powder could not be evaluated since only a single high exposure level was used by Kong et al. According to ECHA endpoint-specific guidance (ECHA, 2015), the Kong et al. results for metallic nickel (single dose) would not be sufficient to fulfil information requirements for metallic nickel or for the purposes of classification and risk assessment. Yet when the new data with micron-size nickel metal powders is considered in a weight of evidence approach with the rest of the available information regarding the known effect of nickel ions in rats, it introduces uncertainty about the reproductive potential of micron-size powders and massive forms of nickel metal and calls into question the current adaptation approach to fulfill reproductive toxicity data requirements for the registered nickel metal materials (micron-size power and massive forms of nickel metal, not including nickel metal nano-particulate forms). For these reasons, EOGRTS testing with nickel metal micron-size powder is proposed (See attached testing proposal).

It should be noted that the current workplace inhalation DNEL for nickel metal (inhalable 0.05 mg Ni/m3) based on protecting workers from possible respiratory toxicity effects is the same value as the DNEL derived for soluble nickel compounds (inhalable 0.05 Ni/m3), which is based on protecting workers from possible respiratory toxicity and carcinogenicity effects as well as from possible reproductive effects (See Section 2.7 of Appendix C2 to the nickel metal CSR). Since the DNEL value is protective of possible reproductive effects with the most bioavailable of the Ni substances, this value is protective of possible effects associated with exposure to metal nickel particles.Therefore, the risk management measures that are already put in place would manage the risks being explored by the proposed testing (Annex I, 0.5 REACH).

Proposed Testing

In view of the uncertainty introduced by the recent reproductive study (Kong et al., 2014) which included a single dose level of nickel metal micron-size powders (as discussed above), and to fulfil Annex X data requirements under REACH, an EOGRTS with micron-sized nickel metal in rats is proposed. An EOGRTS and not a PNDT is selected to cover the most sensitive type of developmental toxicity effects in rats that need to be clarified (i.e. perinatal toxicity) regarding nickel metal, as there are already two PNDT tests available with soluble nickel compounds in two species in addition to the large human reproductive studies with nickel workers discussed above.

Application of Testing Results

The conduct of an EOGRTS with micron-size nickel metal is intended to fulfill data requirements for reproductive developmental toxicity under REACH. In addition, the generated animal data can be considered together with the human data to determine the classification for nickel metal micron powders. If perinatal mortality (or other reproductive effects) are observed that meet classification criteria, nickel metal will be classified for reproductive (developmental) toxicity and a nickel-metal-specific DNEL for these effects will be derived (which will be higher than the current DNEL based on respiratory toxicity effects, due to the expected lower or, at worst-case, equal oral absorption of Ni ion from micron-size nickel metal powder compared to water-soluble nickel compounds). If no reproductive effects are observed in the study, no changes to the current classification would be needed but data fulfillment requirements would have been met. In the meantime, it is important to note that the current workplace DNEL for nickel metal is considered to be protective of any possible reproductive effects associated with nickel-containing substances, and all appropriate risk management measures are in place.

Proposed Route of Administration

REACH specifies that reproductive toxicity studies should be conducted via the “most appropriate route of administration, having regard to the likely route of human exposure”. Exposure pathways for metallic Ni are quite variable between occupationally exposed professionals and consumers, with the primary source of exposure to nickel metal particles being via inhalation for workers during nickel metal production and use (e.g., alloy production, spraying, and grinding). In contrast, the general public is predominantly exposed to nickel metal dermally or orally. Dermal exposure to massive forms of nickel metal and alloys (e.g.in architectural materials) do not lead to significant systemic nickel absorption (dermal absorption~0.2%). While food contact materials containing massive forms of nickel metal and can lead to ingestion of leached Ni ions by the general public, these levels are low (compared to intake of naturally occurring nickel in foods) due to regulatory migration limits in place for food contact materials (see the Exposure section of the nickel metal REACH CSR for more details). Therefore, the work environment is of primary concern.

Regarding inhalation in the workplace, most inhaled particles are large enough to be deposited in the upper respiratory tract from where they are swallowed and subject to absorption from the gastrointestinal tract. This partially accounts for ECHA’s endpoint-specific guidance which recommends the testing of particulates via oral exposure (ECHA, 2015). Additionally, since the oral route would maximize absorption and systemic bioavailability of nickel ions from nickel metal (based on bioelution tests in various physiological fluids), the oral route (by gavage) is proposed to be used in this study. Nickel metal powder will be suspended in distilled water with 0.5% carboxymethylcellulose (CMC), a biologically unreactive material to maintain particles in suspension (Fritz and Becker, 1981), and administered by gavage. Gavage is chosen over dietary delivery because it is the dosing method used by Kong et al. (2014), who reported perinatal mortality effects after in a single group of rats exposed to a high-dose of nickel metal micron particles. Administration by gavage maximizes systemic exposure to test material, allows more precise dosing, and avoids any palatability and subsequent food-avoidance issues that high levels of nickel metal powder in food could cause. The proposed testing will be conducted in rats, as they are the preferred species for this test. The strain will be selected in conjunction with the laboratory to ensure that sufficient, comprehensive, and consistent historical control data are available for the rat strain used in the study. The number of animals will follow the recommendation in the OECD 443 guideline. 

Alternatives to Vertebrate Testing Previously Considered

Before testing was proposed, adaptation possibilities to the testing requirement were explored to fulfill this endpoint. Those include:

1.                  Available GLP and non-GLP studies, and historical human data: As discussed above, there are no K1/K2 or OECD guideline-compliant studies available to meet the testing requirements for reproductive toxicity for micron-size metallic nickel powder. The Kong et al. (2014) study is considered as ‘K3’ for reliability with regards to micron nickel metal powder due to the use of a single dose level. Human data have not demonstrated adverse reproductive effects in well-conducted studies for a female cohort exposed to nickel metal powders in the presence of exposure to other chemical forms of nickel, but EOGRT testing is needed to clarify the potential hazard.

2.                  QSAR: QSAR approaches are not applicable to inorganic metal substances and/or not sufficient to evaluate the reproductive toxicity potential of nickel metal. While the bioavailable Ni ion is responsible for the reproductive effects of soluble nickel compounds in rats, additional data on thein vivobioavailability of nickel from registered nickel metal (as provided by an EOGRTS) is needed to assess its reproductive potential. An EOGRTS is proposed over just toxicokinetic studies as it will provide more definitive data without significantly increasing the total number of animals required for testing. Further, even if a toxicokinetic study were conducted first, there is a high probability that an EOGRTS study will be needed to definitively assess the reproductive toxicity of nickel metal and fulfill REACH data requirements.

3.                  In vitro methods: In vitro methods to replace such a complex test design of an extended one-generation reproductive toxicity study are not available. Bioelution test results in gastric fluid can provide information on relative release of metal ion but cannot provide an in vivo “threshold” value below which no reproductive effects are expected.

4.                  Grouping and read-across: As described above, until now, the Ishimatsu et al. (1995) data has been considered as evidence of very low bioavailability of nickel from oral intake of nickel metal micron-size powders. The recent Kong et al. (2014) study questioned that premise and indicates that currently available data is not sufficient to perform an unequivocal read across to fulfil the data requirements for this endpoint. Importantly, the Kong et al. study does not provide sufficient certainty in itself, as the testing performed with micron-size nickel metal does not meet the criterial for a reliability score of K2 or above.

Consideration was also given to the specific adaptation possibilities of Annex X, 8.7 (and column 2 thereof), and they were found to be not applicable. Nickel metal is not known to be a genotoxic carcinogen or germ cell mutagen, and cannot be considered to have low toxicological activity, lack systemic absorption, or have low or no significant human exposure. Therefore, proposing an EOGRTS for reproductive toxicity of micron-size nickel metal powder is appropriate.

EOGRTS-Specific Study Design Considerations

The following study design variables will be addressed below:

1.                  the need to determine the pre-mating exposure duration (two vs. ten weeks)

2.                  the need to extend Cohort 1B and termination time for F2 generation

3.                  the need to include Cohorts 2A and 2B (developmental neurotoxicity cohorts)

4.                  the need to include Cohort 3 (developmental immunotoxicity cohort).

Premating exposure duration

The OECD TG 443 for EOGRTS indicates that a 2-week premating treatment for both sexes is considered adequate in most cases, as this period covers 3-4 complete estrous cycles in females, and will result in exposure for at least one entire spermatogenic process at the time of termination, when testicular and epidydimal histopathology and analysis of sperm parameters are scheduled. However, ECHA endpoint-specific guidance recommends that the starting point for deciding on the length of premating exposure period should be 10 weeks to adequately assess the fertility endpoint in chemicals with no other information available (ECHA, 2015). As described above, 13-week and one- and two-generation studies utilizing inhalation or oral administration of water-soluble nickel compounds (with the highest bioavailability) have not indicated adverse effects on fertility, estrous cycling, sperm parameters, vaginal cytology, copulation and fertility indices, precoital intervals, gestation lengths, or gross necropsy findings and histopathology of reproductive organs (Ambrose et al., 1976; Smith et al., 1993; RTI, 1988a,b; NTP, 1996; SLI 2000a,b). The only effect observed in previous studies, and the only resulting classification of (soluble) nickel compounds, applies specifically for developmental effects (i.e., perinatal mortality) observed in the rat studies (Ambrose et al., 1976; Smith et al., 1993; SLI 2000a,b). Even the recent Kong et al. (2014) study with nano-size nickel metal particles (and also covering to a limited extent micron-size nickel metal particles) that prompted the submission of this testing proposal did not observe fertility effects. Based on the complete absence of adverse effects on fertility in previous studies using nickel metal or nickel compounds with higher bioavailability than nickel metal, an extension of the pre-mating exposure to 10 weeks is not considered necessary. Finally, a 2-week exposure is supported by animal welfare considerations as it reduces the amount of time the animals are exposed to the test compound at toxic levels, lessening animal suffering. Consequently, the standard pre-mating exposure duration of 2 weeks as outlined in the OECD TG 443 is proposed.

Extension of cohort 1B and termination time for F2 generation

REACH Annex X, 8.7.3, Col. 2, defines conditions under which the F2 Generation shall be produced, which are:

(a) the substance has uses leading to significant exposure of consumers or professionals, taking into account, inter alia, consumer exposure from articles, and

(b) any of the following conditions are met:

— the substance displays genotoxic effects in somatic cell mutagenicity tests in vivo which could lead to classifying it as Mutagen Category 2, or

— there are indications that the internal dose for the substance and/or any of its metabolites will reach a steady state in the test animals only after an extended exposure, or

— there are indications of one or more relevant modes of action related to endocrine disruption from available in vivo studies or non-animal approaches.

The conditions for the production of the F2 Generation do not apply to the test substance. Data regarding carcinogenicity and genotoxic effects of nickel metal do not support a need to classify for the mutagenicity endpoint while mutation studies are ongoing; there are no indications that an extended exposure will be necessary for the internal dose of nickel metal to reach steady state since a one-generation study showed adverse effects with one dose of nickel metal micron-size powder (Kong et al., 2014); and there are no indications that relevant modes of action of nickel metal are related to endocrine disruption. In addition, in previous rat studies with soluble nickel compounds (with higher bioavailability than the test substance) developmental effects (e.g.,perinatal mortality) were identified in the first generation, and there have been no instances of the effects being amplified in the second generation (Ambrose et al., 1976; 1993; RTI, 1988a,b; SLI 2000b). Therefore, even though exposure to professionals and consumers exist, the proposed study design does not foresee the extension of cohort 1B to produce the F2 generation based on the criteria listed above.

Inclusion of cohorts 2A and 2B (developmental neurotoxicity cohorts)

REACH Annex X, 8.7.3, Col. 2, defines conditions under which cohorts 2A and 2B (developmental neurotoxicity cohorts) are to be included, which are:

An Extended One-Generation Reproductive Toxicity Study including cohorts 2A/2B (developmental neurotoxicity) shall be proposed by the registrant or may be required by the Agency in accordance with Article 40 or 41, in case of particular concerns on (developmental) neurotoxicity justified by any of the following:

— existing information on the substance itself derived from relevant available in vivo or non-animal approaches (e.g.abnormalities of the CNS, evidence of adverse effects on the nervous system in studies on adult animals or animals exposed prenatally), or

— specific mechanisms/modes of action of the substance with an association to (developmental) neurotoxicity (e.g.cholinesterase inhibition), or

— existing information on effects caused by substances structurally analogous to the substance being studied, suggesting such effects or mechanisms/modes of action.

The conditions for the inclusion of these cohorts do not apply to the test substance. In previous 28-day, 90-day, and 2-year inhalation studies with micron-size nickel metal powder, and in 90-day and 2-year oral studies with soluble nickel (with higher bioavailability), no histological alterations were observed in the central nervous system (Ambrose et al., 1976; Smith et al., 1993; RTI 1988a,b; NTP 1996; SLI 2000a,b.). Furthermore, no behavioral effects were observed that were not considered to be secondary to general toxicity (WIL Research, 2002, 2004; Oller et al., 2008; Heim et al., 2007). Therefore, the proposed study design does not foresee the addition of the cohorts 2A and 2B (developmental neurotoxicity cohorts).

Inclusion of cohort 3 (developmental immunotoxicity cohort)

REACH Annex X, 8.7.3, Col. 2, indicates particular concerns that would justify the inclusion of cohort 3 (developmental immunotoxicity cohort), which are: 

An Extended One-Generation Reproductive Toxicity Study including cohort 3 (developmental immunotoxicity) shall be proposed by the registrant or may be required by the Agency in accordance with Article 40 or 41, in case of particular concerns on (developmental) immunotoxicity justified by any of the following:

— existing information on the substance itself derived from relevant available in vivo or non-animal approaches (e.g.evidence of adverse effects on the immune system in studies on adult animals or animals exposed prenatally), or

— specific mechanisms/modes of action of the substance with an association to (developmental) immunotoxicity (e.g.relevant changes in thyroidal hormone levels associated to adverse effects), or

— existing information on effects caused by substances structurally analogous to the substance being studied, suggesting such effects or mechanisms/modes of action.

Existing data on the test substance do not indicate those concerns are relevant in this case. In 28-day, 90-day and 2-year studies with nickel metal inhalation in rodents, no severe statistically and/or biologically significant organ weight (spleen, thymus) or histopathological finding related to an immunology organ has been observed (WIL Research, 2002, 2004; Oller et al., 2008). Increases in erythropoiesis (e.g.elevation of red blood cell levels, hemoglobin, and hematocrit values) were observed in rats exposed to nickel metal by inhalation for 12 or 18 months (Oller et al., 2008). There was also accumulation of iron-positive pigment in the kidneys and evidence of extramedullary hematopoiesis in the spleen of exposed animals. These effects are considered primarily due to the hypoxia that ensues when the lung toxicity (resulting from inhalation of nickel metal powder) impairs proper gas exchange (Oller et al., 2008). Increased erythropoietin production and secondary increases in red blood cells are known to occur with chronic pulmonary disease (Boorman and Eustis, 1990). Similar but milder effects on red blood cell numbers, hematocrit, and/or hemoglobin were observed in an oral study with soluble nickel (with higher bioavailability) in which no lung toxicity was observed (Heim et al., 2007). However, in the oral study, the blood changes were small and did not follow a consistent exposure-related pattern. Therefore, the data from both the inhalation and oral studies suggest that most of the observed changes in hematopoiesis in the nickel metal study are secondary to the toxicity effects in the lung.

Inhalation of nickel metal particles has been shown to increase the number of macrophages and polymorphonuclear cells (i.e.,neutrophils) in bronchoalveolar lavage fluid at exposure levels causing lung inflammation, which is a general reaction to any inhaled particles. Therefore, impairment of particle clearance or suppression of T-cell function was not observed. An immunotoxicity study by Graham et al. (1978) involved the inhalation of a soluble nickel compound for 2 hours followed by the determination of the number of antibody-producing spleen cells. The NOAEC for a reduction in the number of antibody-producing cells identified in this study is higher than the NOAEC for respiratory effects after repeated exposure to soluble Ni compounds. That lower value is used in the derivation of the inhalation DNEL. Therefore, the current inhalation DNEL is protective for any such immunotoxic effect. While nickel metal and all nickel compounds are classified as moderate/medium potency dermal sensitizers, the dermal DNEL has been derived to protect from these effects. Lastly, there is no evidence for a hormonal mode of action for immunotoxicity of nickel.  Consequently, the proposed study design does not foresee the addition of the cohort 3 (developmental immunotoxicity cohort), as the criteria for inclusion is not met.


[1]Background urinary nickel levels in female Monchegorsk population had a geometric mean of 5.9 µg/l, the low exposure refinery workers had urinary levels up to 70 µg/l (~12-fold increase in urinary levels) and the high exposure workers had urinary levels between 70 and 179 µg /l (up to 30-fold increase in urinary levels). Urinary nickel levels are better indicators of fetal exposure, as they account for systemically absorbed nickel ion from occupational and non-occupational sources (e.g.diet) by all routes of exposure.

[2]The blood nickel levels achieved in the lifetime inhalation exposure study at the maximum tolerated dose (Oller et al., 2008) are several-fold lower than those reached in a lifetime oral (gavage) study with nickel sulfate in Fischer 344 rats (Heim et al., 2007).

[3]Systemic nickel absorption via the dermal route for nickel metal powder is very low (0.2%) (Hostynek et al., 2001).

[4]Nano-size particles are not included in the nickel metal registration dossier.

Toxicity to reproduction: other studies

Description of key information

A summary document is attached in sections 7.8.1, 7.8.2, and 7.10.2 of IUCLID and inAppendix B6of this CSR discussing human assessments of reproductive impairment associated with soluble nickel exposures as a worst case scenario. In summary, a reproductive study of female refinery workers in Russia has not demonstrated an association between relatively high soluble nickel exposures (worst case scenario with higher blood and urinary levels) and the following reproductive outcomes: genital malformations (hypospadias and cryptorchidism), spontaneous abortions, small-for-gestational-age newborns, and skeletal malformations (Vaktskjold et al., 2006, 2007, 2008a&b).

Additional information

A reproductive study of female refinery workers in Russia has not demonstrated an association between relatively high soluble nickel exposures (worst case scenario for exposure to all nickel substances including nickel metal as it results in higher blood and urinary levels) and the following reproductive outcomes: genital malformations (hypospadias and cryptorchidism), spontaneous abortions, small-for-gestational-age newborns, and skeletal malformations (Vaktskjold et al., 2006, 2007, 2008a&b). Researchers created a birth registry for all births occurring in the region of a Russian nickel refinery at Monchegorsk during the period of the study, which included information on 22,836 newborns and 2,793 pregnancy outcomes surveyed for spontaneous abortions (Vaktskjoldet al.2007; 2008a). They also reconstructed the exposures (using air and urinary nickel measurements) for the female workers at the refineries so as to be able to link specific pregnancy outcomes with occupational exposures via inhalation and systemically bioavailable doses of nickel (i.e.urinary nickel levels). The study culminated in a series of manuscripts by Vaktskjoldet al.(2006, 2007, 2008a,b) describing the results of the investigation. The study demonstrated nickel compound and metal exposure was not associated with adverse pregnancy outcome for 1) male newborns with genital malformations, 2) spontaneous abortions, 3) small-for-gestational-age newborns, or 4) musculoskeletal effects in newborns of female refinery workers exposed to nickel. The data in these manuscripts showed no correlation between nickel exposures (urinary levels as high as 30-fold over background[1]) and observed reproductive impairment. It is important to note thatgenital malformations are considered as one of the most sensitive endpoints for human developmental toxicity while spontaneous abortion in humans would most closely approximate the observation of perinatal lethality associated with nickel exposure in rodents. Further evidence that nickel compound and metal exposure was not adversely affecting the reproduction of these women was provided by the lack of a “small-for-gestational-age” finding and also the lack of an association of male genital malformations with nickel exposure. Both of these findings are considered “sentinel” effects (i.e., sensitive endpoints) for reproductive toxicity in humans.

 

The full document summarizing these data in humans is attached in Sections 7.8 and 7.10.2 of IUCLID and inAppendix B6to the CSR.


[1]Background urinary nickel levels in female Monchegorsk population had a geometric mean of 5.9 µg/l, the low exposure refinery workers had urinary levels up to 70 µg/l (~12-fold increase in urinary levels) and the high exposure workers had urinary levels between 70 and 179 µg /l (up to 30-fold increase in urinary levels). Urinary nickel levels are better indicators of fetal exposure, as they account for systemically absorbed nickel ion from occupational and non-occupational sources (e.g. diet) by all routes of exposure.

Justification for classification or non-classification

Human epidemiological data have not demonstrated a reproductive hazard for nickel exposure. However, in view of the uncertainty introduced by the recent reproductive study (Konget al., 2014) which included a single dose level of nickel metal micron-size powders (as discussed above), and to fulfill Annex X data requirements under REACH, an EOGRTS with micron-sized nickel metal in rats is proposed. An EOGRTS and not a PNDT is selected to cover the most sensitive type of developmental toxicity effects in rats that need to be clarified (i.e.perinatal toxicity) regarding nickel metal, as there are already two PNDT tests available in two species with a soluble nickel compound along with toxicokinetic data to inform their applicabity to nickel metal, in addition to the large human reproductive studies with nickel workers discussed above. Please see ECHA website for the complete EOGRTS testing proposal.